Time-resolved single-cell transcriptomics defines immune trajectories in glioblastoma.

Deciphering the cell-state transitions underlying immune adaptation across time is fundamental for advancing biology. Empirical in vivo genomic technologies that capture cellular dynamics are currently lacking. We present Zman-seq, a single-cell technology recording transcriptomic dynamics across time by introducing time stamps into circulating immune cells, tracking them in tissues for days. Applying Zman-seq resolved cell-state and molecular trajectories of the dysfunctional immune microenvironment in glioblastoma. Within 24 hours of tumor infiltration, cytotoxic natural killer cells transitioned to a dysfunctional program regulated by TGFB1 signaling. Infiltrating monocytes differentiated into immunosuppressive macrophages, characterized by the upregulation of suppressive myeloid checkpoints Trem2, Il18bp, and Arg1, over 36 to 48 hours. Treatment with an antagonistic anti-TREM2 antibody reshaped the tumor microenvironment by redirecting the monocyte trajectory toward pro-inflammatory macrophages. Zman-seq is a broadly applicable technology, enabling empirical measurements of differentiation trajectories, which can enhance the development of more efficacious immunotherapies.

Dynamic mapping of proteome trafficking within and between living cells by TransitID.

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

A pan-cancer single-cell transcriptional atlas of tumor infiltrating myeloid cells.

Tumor-infiltrating myeloid cells (TIMs) are key regulators in tumor progression, but the similarity and distinction of their fundamental properties across different tumors remain elusive. Here, by performing a pan-cancer analysis of single myeloid cells from 210 patients across 15 human cancer types, we identified distinct features of TIMs across cancer types. Mast cells in nasopharyngeal cancer were found to be associated with better prognosis and exhibited an anti-tumor phenotype with a high ratio of TNF+/VEGFA+ cells. Systematic comparison between cDC1- and cDC2-derived LAMP3+ cDCs revealed their differences in transcription factors and external stimulus. Additionally, pro-angiogenic tumor-associated macrophages (TAMs) were characterized with diverse markers across different cancer types, and the composition of TIMs appeared to be associated with certain features of somatic mutations and gene expressions. Our results provide a systematic view of the highly heterogeneous TIMs and suggest future avenues for rational, targeted immunotherapies.

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.

Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

Onco-fetal Reprogramming of Endothelial Cells Drives Immunosuppressive Macrophages in Hepatocellular Carcinoma.

We employed scRNA sequencing to extensively characterize the cellular landscape of human liver from development to disease. Analysis of ∼212,000 cells representing human fetal, hepatocellular carcinoma (HCC), and mouse liver revealed remarkable fetal-like reprogramming of the tumor microenvironment. Specifically, the HCC ecosystem displayed features reminiscent of fetal development, including re-emergence of fetal-associated endothelial cells (PLVAP/VEGFR2) and fetal-like (FOLR2) tumor-associated macrophages. In a cross-species comparative analysis, we discovered remarkable similarity between mouse embryonic, fetal-liver, and tumor macrophages. Spatial transcriptomics further revealed a shared onco-fetal ecosystem between fetal liver and HCC. Furthermore, gene regulatory analysis, spatial transcriptomics, and in vitro functional assays implicated VEGF and NOTCH signaling in maintaining onco-fetal ecosystem. Taken together, we report a shared immunosuppressive onco-fetal ecosystem in fetal liver and HCC. Our results unravel a previously unexplored onco-fetal reprogramming of the tumor ecosystem, provide novel targets for therapeutic interventions in HCC, and open avenues for identifying similar paradigms in other cancers and disease.

Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages.

Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor T cell responses. Jag2-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered T cell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.

Glucose-driven histone lactylation promotes the immunosuppressive activity of monocyte-derived macrophages in glioblastoma.

Immunosuppressive macrophages restrict anti-cancer immunity in glioblastoma (GBM). Here, we studied the contribution of microglia (MGs) and monocyte-derived macrophages (MDMs) to immunosuppression and mechanisms underlying their regulatory function. MDMs outnumbered MGs at late tumor stages and suppressed T cell activity. Molecular and functional analysis identified a population of glycolytic MDM expressing GLUT1 with potent immunosuppressive activity. GBM-derived factors promoted high glycolysis, lactate, and interleukin-10 (IL-10) production in MDMs. Inhibition of glycolysis or lactate production in MDMs impaired IL-10 expression and T cell suppression. Mechanistically, intracellular lactate-driven histone lactylation promoted IL-10 expression, which was required to suppress T cell activity. GLUT1 expression on MDMs was induced downstream of tumor-derived factors that activated the PERK-ATF4 axis. PERK deletion in MDM abrogated histone lactylation, led to the accumulation of intratumoral T cells and tumor growth delay, and, in combination with immunotherapy, blocked GBM progression. Thus, PERK-driven glucose metabolism promotes MDM immunosuppressive activity via histone lactylation.

LC3-Associated Phagocytosis in Myeloid Cells Promotes Tumor Immune Tolerance.

Targeting autophagy in cancer cells and in the tumor microenvironment are current goals of cancer therapy. However, components of canonical autophagy play roles in other biological processes, adding complexity to this goal. One such alternative function of autophagy proteins is LC3-associated phagocytosis (LAP), which functions in phagosome maturation and subsequent signaling events. Here, we show that impairment of LAP in the myeloid compartment, rather than canonical autophagy, induces control of tumor growth by tumor-associated macrophages (TAM) upon phagocytosis of dying tumor cells. Single-cell RNA sequencing (RNA-seq) analysis revealed that defects in LAP induce pro-inflammatory gene expression and trigger STING-mediated type I interferon responses in TAM. We found that the anti-tumor effects of LAP impairment require tumor-infiltrating T cells, dependent upon STING and the type I interferon response. Therefore, autophagy proteins in the myeloid cells of the tumor microenvironment contribute to immune suppression of T lymphocytes by effecting LAP.

ß2-microglobulin triggers NLRP3 inflammasome activation in tumor-associated macrophages to promote multiple myeloma progression.

As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (ß2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed ß2m promotes ß2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1ß (IL-1ß) and IL-18. This process depends on activation of the NLRP3 inflammasome after ß2m accumulation, as macrophages from NLRP3-deficient mice lack efficient ß2m-induced IL-1ß production. Moreover, depletion or silencing of ß2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by ß2m-induced inflammasome signaling. Our results provide mechanistic evidence for ß2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.

Endogenous Glucocorticoid Signaling Regulates CD8+ T Cell Differentiation and Development of Dysfunction in the Tumor Microenvironment.

Identifying signals in the tumor microenvironment (TME) that shape CD8+ T cell phenotype can inform novel therapeutic approaches for cancer. Here, we identified a gradient of increasing glucocorticoid receptor (GR) expression and signaling from naïve to dysfunctional CD8+ tumor-infiltrating lymphocytes (TILs). Conditional deletion of the GR in CD8+ TILs improved effector differentiation, reduced expression of the transcription factor TCF-1, and inhibited the dysfunctional phenotype, culminating in tumor growth inhibition. GR signaling transactivated the expression of multiple checkpoint receptors and promoted the induction of dysfunction-associated genes upon T cell activation. In the TME, monocyte-macrophage lineage cells produced glucocorticoids and genetic ablation of steroidogenesis in these cells as well as localized pharmacologic inhibition of glucocorticoid biosynthesis improved tumor growth control. Active glucocorticoid signaling associated with failure to respond to checkpoint blockade in both preclinical models and melanoma patients. Thus, endogenous steroid hormone signaling in CD8+ TILs promotes dysfunction, with important implications for cancer immunotherapy.

TBK1-Zyxin signaling controls tumor-associated macrophage recruitment to mitigate antitumor immunity.

Mechanical control is fundamental for cellular localization within a tissue, including for tumor-associated macrophages (TAMs). While the innate immune sensing pathways cGAS-STING and RLR-MAVS impact the pathogenesis and therapeutics of malignant diseases, their effects on cell residency and motility remain incompletely understood. Here, we uncovered that TBK1 kinase, activated by cGAS-STING or RLR-MAVS signaling in macrophages, directly phosphorylates and mobilizes Zyxin, a key regulator of actin dynamics. Under pathological conditions and in STING or MAVS signalosomes, TBK1-mediated Zyxin phosphorylation at S143 facilitates rapid recruitment of phospho-Zyxin to focal adhesions, leading to subsequent F-actin reorganization and reduced macrophage migration. Intratumoral STING-TBK1-Zyxin signaling was evident in TAMs and critical in antitumor immunity. Furthermore, myeloid-specific or global disruption of this signaling decreased the population of CD11b+ F4/80+ TAMs and promoted PD-1-mediated antitumor immunotherapy. Thus, our findings identify a new biological function of innate immune sensing pathways by regulating macrophage tissue localization, thus providing insights into context-dependent mitigation of antitumor immunity.

Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

Cancer-Derived Succinate Promotes Macrophage Polarization and Cancer Metastasis via Succinate Receptor.

Macrophages form a major cell population in the tumor microenvironment. They can be activated and polarized into tumor-associated macrophages (TAM) by the tumor-derived soluble molecules to promote tumor progression and metastasis. Here, we used comparative metabolomics coupled with biochemical and animal studies to show that cancer cells release succinate into their microenvironment and activate succinate receptor (SUCNR1) signaling to polarize macrophages into TAM. Furthermore, the results from in vitro and in vivo studies revealed that succinate promotes not only cancer cell migration and invasion but also cancer metastasis. These effects are mediated by SUCNR1-triggered PI3K-hypoxia-inducible factor 1α (HIF-1α) axis. Compared with healthy subjects and tumor-free lung tissues, serum succinate levels and lung cancer SUCNR1 expression were elevated in lung cancer patients, suggesting an important clinical relevance. Collectively, our findings indicate that the secreted tumor-derived succinate belongs to a novel class of cancer progression factors, controlling TAM polarization and promoting tumorigenic signaling.

ADPGK-AS1 long noncoding RNA switches macrophage metabolic and phenotypic state to promote lung cancer growth.

Long noncoding RNAs (lncRNAs) influence the transcription of gene networks in many cell types, but their role in tumor-associated macrophages (TAMs) is still largely unknown. We found that the lncRNA ADPGK-AS1 was substantially upregulated in artificially induced M2-like human macrophages, macrophages exposed to lung cancer cells in vitro, and TAMs from human lung cancer tissue. ADPGK-AS1 is partly located within mitochondria and binds to the mitochondrial ribosomal protein MRPL35. Overexpression of ADPGK-AS1 in macrophages upregulates the tricarboxylic acid cycle and promotes mitochondrial fission, suggesting a phenotypic switch toward an M2-like, tumor-promoting cytokine release profile. Macrophage-specific knockdown of ADPGK-AS1 induces a metabolic and phenotypic switch (as judged by cytokine profile and production of reactive oxygen species) to a pro-inflammatory tumor-suppressive M1-like state, inhibiting lung tumor growth in vitro in tumor cell-macrophage cocultures, ex vivo in human tumor precision-cut lung slices, and in vivo in mice. Silencing ADPGK-AS1 in TAMs may thus offer a novel therapeutic strategy for lung cancer.

Tumor associated macrophage in HPV+ tumors: Between immunosuppression and inflammation.

Over the past few decades, with the rise of immunotherapies, tumor infiltrating immune cells were increasingly investigated. Indeed, they may represent biomarkers for patient outcome prediction, they may bear immune checkpoint markers that can be targeted by therapeutic antibodies and mechanistic studies may reveal how to tweak their activation profile so that we can re-direct them towards tumor cells. Macrophages possess a central place in tissue homeostasis for tissue remodeling and cleaning, transformed cell elimination, phagocytosis and regulation of inflammation via cytokine production. All these functions allow the discovery of approaches to target Tumor Associated Macrophages (TAMs) using immunotherapies. Indeed, TAMs express known immune checkpoint markers such as PD-L1, CD40, Sirp-α and markers such as CD163, CD204, TREM2, TREM1 associated with prognosis. In the context of therapies TAM may participate to antibody dependent cell phagocytosis (ADCP) thanks to FCγ-Receptors. Here, we will review the recent literature on TAMs in the specific context of HPV+ tumors. Indeed, HPV infection of mucosal tissue may lead to head and neck, cervical, penile, anal and vaginal cancers. HPV+ tumors exhibit a higher immune cell infiltrate, which relies on inflammation, immunosuppression and anti-viral response. In this context, and considering the many functions on macrophages, we will show the versatility of TAMs in a tumor microenvironment with viral infection features.

Defining and targeting tumor-associated macrophages in malignant mesothelioma.

Defining the ontogeny of tumor-associated macrophages (TAM) is important to develop therapeutic targets for mesothelioma. We identified two distinct macrophage populations in mouse peritoneal and pleural cavities, the monocyte-derived, small peritoneal/pleural macrophages (SPM), and the tissue-resident large peritoneal/pleural macrophages (LPM). SPM rapidly increased in tumor microenvironment after tumor challenge and contributed to the vast majority of M2-like TAM. The selective depletion of M2-like TAM by conditional deletion of the Dicer1 gene in myeloid cells (D-/-) promoted tumor rejection. Sorted SPM M2-like TAM initiated tumorigenesis in vivo and in vitro, confirming their capacity to support tumor development. The transcriptomic and single-cell RNA sequencing analysis demonstrated that both SPM and LPM contributed to the tumor microenvironment by promoting the IL-2-STAT5 signaling pathway, inflammation, and epithelial-mesenchymal transition. However, while SPM preferentially activated the KRAS and TNF-α/NFkB signaling pathways, LPM activated the IFN-γ response. The importance of LPM in the immune response was confirmed by depleting LPM with intrapleural clodronate liposomes, which abrogated the antitumoral memory immunity. SPM gene signature could be identified in pleural effusion and tumor from patients with untreated mesothelioma. Five genes, TREM2, STAB1, LAIR1, GPNMB, and MARCO, could potentially be specific therapeutic targets. Accordingly, Trem2 gene deletion led to reduced SPM M2-like TAM with compensatory increase in LPM and slower tumor growth. Overall, these experiments demonstrate that SPM M2-like TAM play a key role in mesothelioma development, while LPM more specifically contribute to the immune response. Therefore, selective targeting of monocyte-derived TAM may enhance antitumor immunity through compensatory expansion of tissue-resident TAM.

Tumor progression is independent of tumor-associated macrophages in cell lineage-based mouse models of glioblastoma.

Macrophage targeting therapies have had limited clinical success in glioblastoma (GBM). Further understanding the GBM immune microenvironment is critical for refining immunotherapeutic approaches. Here, we use genetically engineered mouse models and orthotopic transplantation-based GBM models with identical driver mutations and unique cells of origin to examine the role of tumor cell lineage in shaping the immune microenvironment and response to tumor-associated macrophage (TAM) depletion therapy. We show that oligodendrocyte progenitor cell lineage-associated GBMs (Type 2) recruit more immune infiltrates and specifically monocyte-derived macrophages than subventricular zone neural stem cell-associated GBMs (Type 1). We then devise a TAM depletion system that offers a uniquely robust and sustained TAM depletion. We find that extensive TAM depletion in these cell lineage-based GBM models affords no survival benefit. Despite the lack of survival benefit of TAM depletion, we show that Type 1 and Type 2 GBMs have unique molecular responses to TAM depletion. In sum, we demonstrate that GBM cell lineage influences TAM ontogeny and abundance and molecular response to TAM depletion.

Roles of the immune system in cancer: from tumor initiation to metastatic progression.

The presence of inflammatory immune cells in human tumors raises a fundamental question in oncology: How do cancer cells avoid the destruction by immune attack? In principle, tumor development can be controlled by cytotoxic innate and adaptive immune cells; however, as the tumor develops from neoplastic tissue to clinically detectable tumors, cancer cells evolve different mechanisms that mimic peripheral immune tolerance in order to avoid tumoricidal attack. Here, we provide an update of recent accomplishments, unifying concepts, and future challenges to study tumor-associated immune cells, with an emphasis on metastatic carcinomas.