InVivoMAb anti-mouse/human/rat CCL2 (MCP-1)

Immunotherapy represents a pivotal strategy for triple-negative breast cancer (TNBC), yet its efficacy is constrained by the immunosuppressive tumor microenvironment (TME). In this study, we demonstrate that bone morphogenetic protein-9 (BMP9) inhibits tumor growth and reprograms the immune TME in orthotopic TNBC models, primarily by attenuating regulatory T cells (Tregs) infiltration. Tregs depletion abrogates si-BMP9-mediated tumor promotion. Mechanistically, BMP9 suppresses CCL2 expression in an exocrine-independent manner to restrict Tregs recruitment. We identify DNA-dependent protein kinase catalytic subunit (PRKDC) as a BMP9-binding transcriptional regulator. The interaction between PRKDC and BMP9 directly impedes CCL2 transcriptional activation by suppressing PRKDC phosphorylation and indirectly suppresses CCL2 expression via NF-κB pathway remodeling. Critically, BMP9 modulation and CCL2 targeting potentiates immunotherapy efficacy without observable toxicity. Our study unveils the BMP9-PRKDC-CCL2 axis as a master regulatory node for TNBC-Tregs crosstalk, providing a strategy to overcome immunotherapy resistance in TNBC.

Cells terminally arrested in the cell cycle that exhibit a distinct secretory phenotype are referred to as senescent. These cells play a complex role during tumor progression; they can inhibit or promote tumor growth depending on disease stage. We developed a mouse model that allows monitoring and selective elimination of cells expressing high levels of the cyclin-dependent kinase inhibitor p16 and interleukin-6. These mice, termed SuSe (suicidal senescence), were crossed with the mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) model of breast cancer. Functional characterization in SuSe/PyMT mice confirmed that depletion of senescent cells (senolysis) in early and late lesions accelerated and decelerated tumor growth and metastasis, respectively. Tumor acceleration was attributed to expansion of immunosuppressive, protumorigenic macrophages. C-C motif chemokine ligand 2 was identified as an autocrine chemokine essential for their recruitment and maintenance. Depletion of these macrophages reversed the effects of senescent cell clearance, rendering senolysis antitumorigenic even at early stages. Our results suggest that targeting immunosuppressive macrophages can preserve the benefits of senolysis while mitigating adverse effects.

Rising obesity rates are closely linked to higher risk of cancer, yet the underlying mechanisms are not fully understood. It is previously reported that fatty acids (FAs) released from cancer-associated adipose tissue enhance hypoxia-inducible factor-1α (HIF-1α) expression in cancer cells, promoting tumor progression. Here, it is elucidated that cancer cells manipulate adjacent adipose tissue by secreting C-C chemokine ligand2 (CCL2) to exploit FAs. Activation of HIF-1α induced by FA influx increases CCL2 expression in cancer cells, which subsequently leads to lipolysis in nearby adipose tissue by activating peroxisome proliferator-activated receptor alpha (PPARα) signaling. This activation in adipose tissue results in the release of FAs into the tumor microenvironment. The increased lipid supply to tumor reactivates the FA/HIF-1α/CCL2 axis in cancer cells, further accelerating tumor growth and CCL2 secretion. This establishes a positive feedback loop between tumor and adjacent adipose tissue, which enhances cancer progression. This crosstalk is validated by using a polydimethylsiloxane-based 3D coculture system and in vivo models. In obese mice, this reciprocal signaling accelerated tumor progression, whereas intra-tumoral injection of CCL2-neutralizing antibody significantly suppressed it. These findings reveal a metabolic circuit for tumor survival and disrupting this interaction may provide promising therapeutic targets, particularly for obese cancer patients.

Glutamate is well-known as metabolite for maintaining the energy and redox homeostasis in cancer, moreover it is also the primary excitatory neurotransmitter in the central nervous system. However, whether glutamatergic signaling can regulate hepatocellular carcinoma (HCC) progression and the specific regulatory mechanisms are unknown. In the present study, we found that glutamate and its receptor NMDAR2B were significantly elevated in HCC patients, which predicts poor prognosis. Glutamate could upregulate CCL2 expression on hepatoma cells and further enhance the capability of tumor cells to recruit tumor-associated macrophages (TAMs). Mechanistically, glutamate could facilitate CCL2 expression through NMDAR pathway by decreasing the expression of EZH2, which regulates the H3K27me3 levels on the CCL2 promoter, rather than affecting DNA methylation. Moreover, inhibiting glutamate pathway with MK801 could significantly delay tumor growth, with reduced TAMs in implanted Hepa1-6 mouse HCC models. Our work suggested that glutamate could induce CCL2 expression to promote TAM infiltration by negatively regulating EZH2 levels in hepatoma cells, which might serve as a potential prognostic marker and a therapeutic target for HCC patients.

Novel therapeutic strategies are needed to improve the efficacy of chimeric antigen receptor (CAR) T cells as a treatment of solid tumors. Multiple tumor microenvironmental factors are thought to contribute to resistance to CAR T-cell therapy in solid tumors, and appropriate model systems to identify and examine these factors using clinically relevant biospecimens are limited. In this study, we examined the activity of B7-H3-directed CAR T cells (B7-H3.CAR-T) using 3D microfluidic cultures of patient-derived organotypic tumor spheroids (PDOTS) and then confirmed the activity of B7-H3.CAR T cells in PDOTS. Although B7-H3 expression in PDOTS was associated with B7-H3.CAR-T sensitivity, mechanistic studies revealed dynamic upregulation of co-inhibitory receptors on CAR T-cells following target cell encounter that led to CAR T-cell dysfunction and limited efficacy against B7-H3-expressing tumors. PD-1 blockade restored CAR T-cell activity in monotypic and organotypic tumor spheroids with improved tumor control and upregulation of effector cytokines. Given the emerging role of TANK-binding kinase 1 (TBK1) as an immune evasion gene, we examined the effect of TBK1 inhibition on CAR T-cell efficacy. Similar to PD-1 blockade, TBK1 inhibition restored CAR T-cell activity in monotypic and organotypic tumor spheroids, prevented CAR T-cell dysfunction, and enhanced CAR T-cell proliferation. Inhibition or deletion of TBK1 also enhanced the sensitivity of cancer cells to immune-mediated killing. Taken together, our results demonstrate the feasibility and utility of ex vivo profiling of CAR T cells using PDOTS and suggest that targeting TBK1 could be used to enhance CAR T-cell efficacy by overcoming tumor-intrinsic and -extrinsic resistance mechanisms.

Hepatocellular carcinoma (HCC) often occurs in the context of fibrosis or cirrhosis. Methylation of histone is an important epigenetic mechanism, but it is unclear whether histone methyltransferases are potent targets for fibrosis-associated HCC therapy. ASH1L, an H3K4 methyltransferase, is found at higher levels in activated hepatic stellate cells (HSCs) and hepatoma cells. To determine the role of ASH1L in vivo, transgenic mice with conditional Ash1l depletion in the hepatocyte cell lineage (Ash1lflox/floxAlbcre) or HSCs (Ash1lflox/floxGFAPcreERT2) are generated, and these mice are challenged in a diethylnitrosamine (DEN)/carbon tetrachloride (CCl4)-induced model of liver fibrosis and HCC. Depleting Ash1l in both hepatocytes and HSCs mitigates hepatic fibrosis and HCC development. Multicolor flow cytometry, bulk, and single-cell transcriptomic sequencing reveal that ASH1L creates an immunosuppressive microenvironment. Mechanically, ASH1L-mediated H3K4me3 modification increases the expression of CCL2 and CSF1, which recruites and polarizes M2-like pro-tumorigenic macrophages. The M2-like macrophages further enhance tumor cell proliferation and suppress CD8+ T cell activation. AS-99, a small molecule inhibitor of ASH1L, demonstrates similar anti-fibrosis and tumor-suppressive effects. Of pathophysiological significance, the increased expression levels of mesenchymal ASH1L and M2 marker CD68 are associated with poor prognosis of HCC. The findings reveal ASH1L as a potential small-molecule therapeutic target against fibrosis-related HCC.

Pathogen encounter can result in epigenetic remodeling that shapes disease caused by heterologous pathogens. Here, we examined innate immune memory in the context of commonly circulating respiratory viruses. Single-cell analyses of airway-resident immune cells in a disease-relevant murine model of SARS-CoV-2 recovery revealed epigenetic reprogramming in alveolar macrophages following infection. Post-COVID-19 human monocytes exhibited similar epigenetic signatures. In airway-resident macrophages, past SARS-CoV-2 infection increased activity of type I interferon (IFN-I)-related transcription factors and epigenetic poising of antiviral genes. Viral pattern recognition and canonical IFN-I signaling were required for the establishment of this innate immune memory and augmented secondary antiviral responses. Antiviral innate immune memory mounted by airway-resident macrophages post-SARS-CoV-2 was necessary and sufficient to ameliorate secondary disease caused by influenza A virus and curtailed hyperinflammatory dysregulation and mortality. Our findings provide insights into antiviral innate immune memory in the airway that may facilitate the development of broadly effective therapeutic strategies.

Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment.

Leukocyte immunoglobulin-like receptor B4 (LILRB4) plays a significant role in regulating immune responses. LILRB4 in microglia might influence the infiltration of peripheral T cells. However, whether and how LILRB4 expression aggravates brain damage after acute ischemic stroke remains unclear. This study investigates the role of LILRB4 in modulating the immune response and its potential protective effects against ischemic brain injury in mice.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

Specific microbial signals induce the differentiation of a distinct pool of RORγ+ regulatory T (Treg) cells crucial for intestinal homeostasis. We discovered highly analogous populations of microbiota-dependent Treg cells that promoted tissue regeneration at extra-gut sites, notably acutely injured skeletal muscle and fatty liver. Inflammatory meditators elicited by tissue damage combined with MHC-class-II-dependent T cell activation to drive the accumulation of gut-derived RORγ+ Treg cells in injured muscle, wherein they regulated the dynamics and tenor of early inflammation and helped balance the proliferation vs. differentiation of local stem cells. Reining in IL-17A-producing T cells was a major mechanism underlying the rheostatic functions of RORγ+ Treg cells in compromised tissues. Our findings highlight the importance of gut-trained Treg cell emissaries in controlling the response to sterile injury of non-mucosal tissues.

Oncogenic mutations in PIK3CA, encoding p110α-PI3K, are a common cause of venous and lymphatic malformations. Vessel type-specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit pro-lymphangiogenic macrophages to promote progressive lymphatic overgrowth. Mouse model of Pik3caH1047R-driven vascular malformations showed that proliferation was induced in both venous and lymphatic ECs but sustained selectively in LECs of advanced lesions. Single-cell transcriptomics identified the iLEC population, residing at lymphatic capillary terminals of normal vasculature, that was expanded in Pik3caH1047R mice. Expression of pro-inflammatory genes, including monocyte/macrophage chemokine Ccl2, in Pik3caH1047R-iLECs was associated with recruitment of VEGF-C-producing macrophages. Macrophage depletion, CCL2 blockade, or anti-inflammatory COX-2 inhibition limited Pik3caH1047R-driven lymphangiogenesis. Thus, targeting the paracrine crosstalk involving iLECs and macrophages provides a new therapeutic opportunity for lymphatic malformations. Identification of iLECs further indicates that peripheral lymphatic vessels not only respond to but also actively orchestrate inflammatory processes.

Complex communications between hepatocytes and Kupffer cells (KCs) are known to drive or suppress hepatocarcinogenesis, with controversial data in the literature. In previous experiments that aimed to decipher hepatocyte/KC interactions, we unexpectedly unveiled a tumor-suppressing effect of polyinosinic-polycytidylic acid, a widely used inducer of MX dynamin like GTPase 1 (Mx1)-cre expression, which questioned a theory of interleukin 1a/6 cytokine circuit in hepatocyte/KC communication. The goal of this study was to clarify the controversy and decipher unique functions of KCs and non-KC macrophages in liver tumorigenesis.

Although aging enhances atherosclerosis, we do not know if this occurs via alterations in circulating immune cells, lipid metabolism, vasculature, or adipose tissue. Here, we examined whether aging exerts a direct pro-atherogenic effect on adipose tissue in mice. After demonstrating that aging augmented the inflammatory profile of visceral but not subcutaneous adipose tissue, we transplanted visceral fat from young or aged mice onto the right carotid artery of Ldlr-/- recipients. Aged fat transplants not only increased atherosclerotic plaque size with increased macrophage numbers in the adjacent carotid artery, but also in distal vascular territories, indicating that aging of the adipose tissue enhances atherosclerosis via secreted factors. By depleting macrophages from the visceral fat, we identified that adipose tissue macrophages are major contributors of the secreted factors. To identify these inflammatory factors, we found that aged fat transplants secreted increased levels of the inflammatory mediators TNFα, CXCL2, and CCL2, which synergized to promote monocyte chemotaxis. Importantly, the combined blockade of these inflammatory mediators impeded the ability of aged fat transplants to enhance atherosclerosis. In conclusion, our study reveals that aging enhances atherosclerosis via increased inflammation of visceral fat. Our study suggests that future therapies targeting the visceral fat may reduce atherosclerosis disease burden in the expanding older population.

Individuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI).  METHODS: The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1β (IL-1β), and C-C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM.  RESULTS: Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1β signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1β, and CCL2 neutralization, or ICS treatment.

Malignant brain tumours are the cause of a disproportionate level of morbidity and mortality among cancer patients, an unfortunate statistic that has remained constant for decades. Despite considerable advances in the molecular characterization of these tumours, targeting the cancer cells has yet to produce significant advances in treatment. An alternative strategy is to target cells in the glioblastoma microenvironment, such as tumour-associated astrocytes. Astrocytes control multiple processes in health and disease, ranging from maintaining the brain's metabolic homeostasis, to modulating neuroinflammation. However, their role in glioblastoma pathogenicity is not well understood. Here we report that depletion of reactive astrocytes regresses glioblastoma and prolongs mouse survival. Analysis of the tumour-associated astrocyte translatome revealed astrocytes initiate transcriptional programmes that shape the immune and metabolic compartments in the glioma microenvironment. Specifically, their expression of CCL2 and CSF1 governs the recruitment of tumour-associated macrophages and promotes a pro-tumourigenic macrophage phenotype. Concomitantly, we demonstrate that astrocyte-derived cholesterol is key to glioma cell survival, and that targeting astrocytic cholesterol efflux, via ABCA1, halts tumour progression. In summary, astrocytes control glioblastoma pathogenicity by reprogramming the immunological properties of the tumour microenvironment and supporting the non-oncogenic metabolic dependency of glioblastoma on cholesterol. These findings suggest that targeting astrocyte immunometabolic signalling may be useful in treating this uniformly lethal brain tumour.

Neuroinflammation exacerbates the progression of SOD1-driven amyotrophic lateral sclerosis (ALS), although the underlying mechanisms remain largely unknown. Herein, we demonstrate that misfolded SOD1 (SOD1Mut)-causing ALS results in mitochondrial damage, thus triggering the release of mtDNA and an RNA:DNA hybrid into the cytosol in an mPTP-independent manner to activate IRF3- and IFNAR-dependent type I interferon (IFN-I) and interferon-stimulating genes. The neuronal hyper-IFN-I and pro-inflammatory responses triggered in ALS-SOD1Mut were sufficiently robust to cause a strong physiological outcome in vitro and in vivo. cGAS/DDX41-STING-signaling is amplified in bystander cells through inter-neuronal gap junctions. Our results highlight the importance of a common DNA-sensing pathway between SOD1 and TDP-43 in influencing the progression of ALS.

Immunotherapy in colorectal cancer (CRC) regulates specific immune checkpoints and, when used in combination with chemotherapy, can improve patient prognosis. One specific immune checkpoint is the recruitment of circulating monocytes that differentiate into tumor-associated macrophages (TAMs) and promote tumor angiogenesis. Changes in vascularization can be non-invasively assessed via diffuse reflectance spectroscopy using hemoglobin concentrations and oxygenation in a localized tumor volume. In this study, we examine whether blockade of monocyte recruitment via CCL2 (macrophage chemoattractant protein-1) leads to enhanced sensitivity of 5-fluorouracil (5-FU) in a CT26-Balb/c mouse model of CRC. It was hypothesized that the blockade of TAMs will alter tumor perfusion, increasing chemotherapy response. A subcutaneous tumor model using Balb/c mice injected with CT26 colon carcinoma cells received either a saline or isotype control, anti-CCL2, 5-FU, or a combination of anti-CCL2 and 5-FU.

Brain tumor formation and progression are dictated by cooperative interactions between neoplastic and non-neoplastic cells. This stromal dependence is nicely illustrated by tumors arising in the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome, where children develop low-grade optic pathway gliomas (OPGs). Using several authenticated Nf1-OPG murine models, we previously demonstrated that murine Nf1-OPG growth is regulated by T cell function and microglia Ccl5 production, such that their inhibition reduces tumor proliferation in vivo. While these interactions are critical for established Nf1-OPG tumor growth, their importance in tumor formation has not been explored.