LYVE-1+ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer.

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular "nest" structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1+ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1+ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8+ T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy.

Proof-of-concept trial of Goal Management Training+ to improve executive functions and treatment outcomes in methamphetamine use disorder.

BACKGROUND: Deficits in executive function are common in methamphetamine use disorder (MUD), likely contributing to difficulties in sustained treatment success. Cognitive remediation interventions are designed to treat such deficits but have not been adapted to the needs of people with MUD. This study presents a proof-of-concept trial to evaluate a new cognitive remediation program for MUD, Goal Management Training+ (GMT+). METHODS: This was a cluster-randomised crossover trial comparing GMT+ with a psychoeducation-based control (Brain Health Workshop; BHW). GMT+ is a therapist-administered group-based cognitive remediation for executive dysfunction comprising four 90-minute weekly sessions and daily journal activities. BHW is a lifestyle psychoeducation program matched to GMT+ for therapist involvement, format, and duration. Participants (n = 36; GMT n = 17; BHW n = 19) were recruited from therapeutic communities in Victoria, Australia. Primary outcomes included intervention acceptability, feasibility, and improvements in self-reported executive function. Secondary outcomes included cognitive tests of executive function, severity of methamphetamine dependence, craving, and quality of life. We performed mixed linear modelling and calculated Hedges' g effect sizes. RESULTS: GMT+ participant ratings and program retention indicated high acceptability. There was no difference between GMT+ and BHW on self-reported executive function (g = 0.06). Cognitive tasks suggested benefits of GMT+ on information gathering (g = 0.88) and delay-discounting (g = 0.80). Severity of methamphetamine dependence decreased more in GMT+ (g = 1.47). CONCLUSIONS: GMT+ was well-accepted but did not improve self-reported executive functioning. Secondary outcomes suggested GMT+ was beneficial for objective cognitive performance and severity of dependence.

Meningeal macrophages protect against viral neuroinfection.

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II- MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation.

The emerging roles of interstitial macrophages in pulmonary fibrosis: A perspective from scRNA-seq analyses.

Pulmonary fibrosis is an irreversible and progressive disease affecting the lungs, and the etiology remains poorly understood. This disease can be lethal and currently has no specific clinical therapeutic regimen. Macrophages, the most common type of immune cell in the lungs, have been reported to play a key role in the pathogenesis of fibrotic disease. The lung macrophage population is mostly composed of alveolar macrophages and interstitial macrophages, both of which have not been thoroughly studied in the pathogenesis of lung fibrosis. Interstitial macrophages have recently been recognised for their participation in lung fibrosis due to new technology arising from a combination of bioinformatics and single-cell RNA sequencing analysis. This paper reviews recent developments regarding lung macrophage classification and summarizes the origin and replenishment of interstitial macrophages and their function in pulmonary fibrosis.

A novel ZsGreen knock-in melanoma cell line reveals the function of CD11b in tumor phagocytosis.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is an efficient tool for establishing genetic models including cellular models, and has facilitated unprecedented advancements in biomedical research. In both patients and cancer animal models, immune cells infiltrate the tumor microenvironment and some of them migrate to draining lymph nodes to exert antitumor effects. Among these immune cells, phagocytes such as macrophages and dendritic cells engulf tumor antigens prior to their crosstalk with T cells and elicit adaptive immune response against tumors. Melanoma cells are frequently used as a tumor model because of their relatively high level of somatic mutations and antigenicity. However, few genetic models have been developed using melanoma cell lines to track tumor cell phagocytosis, which is essential for understanding protective immune response in vivo. In this study, we used CRISPR/Cas9-mediated DNA cleavage and homologous recombination to develop a novel knock-in tool which expresses the ultra-bright fluorescent probe ZsGreen in YUMM1.7 melanoma cells. Using this novel tool, we measured the macrophagic engulfment of melanoma cells inside the tumor microenvironment. We also found that in tumor-grafted mice, a subset of dendritic cells efficiently engulfed YUMM1.7 cells and was preferentially trafficking tumor antigens to draining lymph nodes. In addition, we used this knock-in tool to assess the impact of a point mutation of CD11b on phagocytosis in the tumor microenvironment. Our results demonstrate that the ZsGreen-expressing YUMM1.7 melanoma model provides a valuable tool for the study of phagocytosis in vivo.

Sympathetic axonal sprouting induces changes in macrophage populations and protects against pancreatic cancer.

Neuronal nerve processes in the tumor microenvironment were highlighted recently. However, the origin of intra-tumoral nerves remains poorly known, in part because of technical difficulties in tracing nerve fibers via conventional histological preparations. Here, we employ three-dimensional (3D) imaging of cleared tissues for a comprehensive analysis of sympathetic innervation in a murine model of pancreatic ductal adenocarcinoma (PDAC). Our results support two independent, but coexisting, mechanisms: passive engulfment of pre-existing sympathetic nerves within tumors plus an active, localized sprouting of axon terminals into non-neoplastic lesions and tumor periphery. Ablation of the innervating sympathetic nerves increases tumor growth and spread. This effect is explained by the observation that sympathectomy increases intratumoral CD163+ macrophage numbers, which contribute to the worse outcome. Altogether, our findings provide insights into the mechanisms by which the sympathetic nervous system exerts cancer-protective properties in a mouse model of PDAC.

CD4 T cell-intrinsic STING signaling controls the differentiation and effector functions of TH1 and TH9 cells.

BACKGROUND: While stimulator of interferon genes (STING) activation in innate immune cells of the tumor microenvironment can result in CD8 T cell-dependent antitumor immunity, whether STING signaling affects CD4 T-cell responses remains elusive. METHODS: Here, we tested whether STING activation modulated the effector functions of CD4 T cells in vivo by analyzing tumor-infiltrating CD4 T cells and evaluating the contribution of the CD4 T cell-derived cytokines in the antitumor activity of the STING ligand 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) in two mouse tumor models. We performed ex vivo experiments to assess the impact of STING activation on CD4 T-cell differentiation and investigate the underlying molecular mechanisms. Finally, we tested whether STING activation enhances TH9 cell antitumor activity against mouse melanoma upon adoptive transfer. RESULTS: We found that activation of STING signaling cell-intrinsically enhances the differentiation and antitumor functions of TH1 and TH9 cells by increasing their respective production of interferon gamma (IFN-γ) and interleukin-9. IRF3 and type I interferon receptors (IFNARs) are required for the STING-driven enhancement of TH1 cell differentiation. However, STING activation favors TH9 cell differentiation independently of the IFNARs/IRF3 pathway but through mammalian target of rapamycin (mTOR) signaling, underscoring that STING activation differentially affects the fate of distinct CD4 T-cell subsets. The therapeutic effect of STING activation relies on TH1 and TH9-derived cytokines, and STING activation enhances the antitumor activity of TH9 cells upon adoptive transfer. CONCLUSION: Our results reveal the STING signaling pathway as a therapeutic target to boost CD4 T-cell effector functions and antitumor immunity.

Macrophages orchestrate the expansion of a proangiogenic perivascular niche during cancer progression.

Tumor-associated macrophages (TAMs) are a highly plastic stromal cell type that support cancer progression. Using single-cell RNA sequencing of TAMs from a spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT), we characterize a subset of these cells expressing lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve-1) that spatially reside proximal to blood vasculature. We demonstrate that Lyve-1+ TAMs support tumor growth and identify a pivotal role for these cells in maintaining a population of perivascular mesenchymal cells that express α-smooth muscle actin and phenotypically resemble pericytes. Using photolabeling techniques, we show that mesenchymal cells maintain their prevalence in the growing tumor through proliferation and uncover a role for Lyve-1+ TAMs in orchestrating a selective platelet-derived growth factor­CC­dependent expansion of the perivascular mesenchymal population, creating a proangiogenic niche. This study highlights the inter-reliance of the immune and nonimmune stromal network that supports cancer progression and provides therapeutic opportunities for tackling the disease.

The Role of Plasmacytoid Dendritic Cells in Cancers.

Plasmacytoid dendritic cells (pDCs) are a special subtype of dendritic cells with the morphology of plasma cells. pDCs produce massive amounts of type I interferon (IFN-I), which was originally found to play an extremely pivotal role in antiviral immunity. Interestingly, accumulated evidence indicates that pDCs can also play an important role in tumorigenesis. In the human body, most of the IFN-α is secreted by activated pDCs mediated by toll-like receptor (TLR) stimulation. In many types of cancer, tumors are infiltrated by a large number of pDCs, however, these pDCs exhibit no response to TLR stimulation, and reduced or absent IFN-α production. In addition, tumor-infiltrating pDCs promote recruitment of regulatory T cells (Tregs) into the tumor microenvironment, leading to immunosuppression and promoting tumor growth. In this review, we discuss recent insights into the development of pDCs and their roles in a variety of malignancies, with special emphasis on the basic mechanisms.

NF-κB-dependent IRF1 activation programs cDC1 dendritic cells to drive antitumor immunity.

Conventional type 1 dendritic cells (cDC1s) are critical for antitumor immunity. They acquire antigens from dying tumor cells and cross-present them to CD8+ T cells, promoting the expansion of tumor-specific cytotoxic T cells. However, the signaling pathways that govern the antitumor functions of cDC1s in immunogenic tumors are poorly understood. Using single-cell transcriptomics to examine the molecular pathways regulating intratumoral cDC1 maturation, we found nuclear factor κB (NF-κB) and interferon (IFN) pathways to be highly enriched in a subset of functionally mature cDC1s. We identified an NF-κB-dependent and IFN-γ-regulated gene network in cDC1s, including cytokines and chemokines specialized in the recruitment and activation of cytotoxic T cells. By mapping the trajectory of intratumoral cDC1 maturation, we demonstrated the dynamic reprogramming of tumor-infiltrating cDC1s by NF-κB and IFN signaling pathways. This maturation process was perturbed by specific inactivation of either NF-κB or IFN regulatory factor 1 (IRF1) in cDC1s, resulting in impaired expression of IFN-γ-responsive genes and consequently a failure to efficiently recruit and activate antitumoral CD8+ T cells. Last, we demonstrate the relevance of these findings to patients with melanoma, showing that activation of the NF-κB/IRF1 axis in association with cDC1s is linked with improved clinical outcome. The NF-κB/IRF1 axis in cDC1s may therefore represent an important focal point for the development of new diagnostic and therapeutic approaches to improve cancer immunotherapy.

An inducible model for specific neutrophil depletion by diphtheria toxin in mice.

Neutrophils are crucial for immunity and play important roles in inflammatory diseases; however, mouse models selectively deficient in neutrophils are limited, and neutrophil-specific diphtheria toxin (DT)-based depletion system has not yet been established. In this study, we generated a novel knock-in mouse model expressing diphtheria toxin receptor (DTR) under control of the endogenous Ly6G promoter. We showed that DTR expression was restricted to Ly6G+ neutrophils and complete depletion of neutrophils could be achieved by DT treatment at 24-48 h intervals. We characterized the effects of specific neutrophil depletion in mice at steady-state, with acute inflammation and during tumor growth. Our study presents a valuable new tool to study the roles of neutrophils in the immune system and during tumor progression.

PAR-1 signaling on macrophages is required for effective in vivo delayed-type hypersensitivity responses.

Delayed-type hypersensitivity (DTH) responses underpin chronic inflammation. Using a model of oxazolone-induced dermatitis and a combination of transgenic mice, adoptive cell transfer, and selective agonists/antagonists against protease activated receptors, we show that that PAR-1 signaling on macrophages by thrombin is required for effective granuloma formation. Using BM-derived macrophages (BMMs) in vitro, we show that thrombin signaling induced (a) downregulation of cell membrane reverse cholesterol transporter ABCA1 and (b) increased expression of IFNγ receptor and enhanced co-localization within increased areas of cholesterol-rich membrane microdomains. These two key phenotypic changes combined to make thrombin-primed BMMs sensitive to M1 polarization by 1000-fold less IFNγ, compared to resting BMMs. We confirm that changes in ABCA1 expression were directly responsible for the exquisite sensitivity to IFNγ in vitro and for the impact on granuloma formation in vivo. These data indicate that PAR-1 signaling plays a hitherto unrecognized and critical role in DTH responses.

Tissue-resident macrophages in omentum promote metastatic spread of ovarian cancer.

Experimental and clinical evidence suggests that tumor-associated macrophages (TAMs) play important roles in cancer progression. Here, we have characterized the ontogeny and function of TAM subsets in a mouse model of metastatic ovarian cancer that is representative for visceral peritoneal metastasis. We show that the omentum is a critical premetastatic niche for development of invasive disease in this model and define a unique subset of CD163+ Tim4+ resident omental macrophages responsible for metastatic spread of ovarian cancer cells. Transcriptomic analysis showed that resident CD163+ Tim4+ omental macrophages were phenotypically distinct and maintained their resident identity during tumor growth. Selective depletion of CD163+ Tim4+ macrophages in omentum using genetic and pharmacological tools prevented tumor progression and metastatic spread of disease. These studies describe a specific role for tissue-resident macrophages in the invasive progression of metastatic ovarian cancer. The molecular pathways of cross-talk between tissue-resident macrophages and disseminated cancer cells may represent new targets to prevent metastasis and disease recurrence.

Non-activatable mutant of inhibitor of kappa B kinase α (IKKα) exerts vascular site-specific effects on atherosclerosis in Apoe-deficient mice.

BACKGROUND AND AIMS: IKKα is an important regulator of gene expression. As IKKα kinase inactivity in bone marrow-derived cells does not affect atherosclerosis, we here investigate the impact of a whole body-IKKα kinase inactivity on atherosclerosis. METHODS: Apolipoprotein E (Apoe)-deficient mice homozygous for an activation-resistant Ikkα-mutant (IkkαAA/AAApoe-/-) and Ikkα+/+Apoe-/- controls received a Western-type diet. Atherosclerotic lesion size and cellular content were analyzed using histology and immunofluorescence. Vascular protein expression and IKKα kinase activity were quantified by Luminex multiplex immuno-assay and ELISA. RESULTS: A vascular site-specific IKKα expression and kinase activation profile was revealed, with higher total IKKα protein levels in aortic root but increased IKKα phosphorylation, representing activated IKKα, in the aortic arch. This was associated with a vascular site-specific effect of IkkαAA/AA knock-in on atherosclerosis: in the aortic root, IkkαAA/AA knock-in decreased lesion size by 22.0 ±â€¯7.7% (p < 0.01), reduced absolute lesional smooth muscle cell numbers and lowered pro-atherogenic MMP2. In contrast, IkkαAA/AA knock-in increased lesion size in the aortic arch by 43.7 ±â€¯20.1% (p < 0.001), increased the abundance of lesional smooth muscle cells in brachiocephalic artery as main arch side branch and elevated MMP2. A similar profile was observed for MMP3. No effects were observed on necrotic core or collagen deposition in atherosclerotic lesions, nor on absolute lesional macrophage numbers. CONCLUSIONS: A non-activatable IKKα kinase differentially affects atherosclerosis in aortic root vs. aortic arch/brachiocephalic artery, associated with a differential vascular IKKα expression and kinase activation profile as well as with a vascular site-dependent impact on lesional smooth muscle cell accumulation and protein expression profiles.

Targeting STAT3 and STAT5 in Tumor-Associated Immune Cells to Improve Immunotherapy.

Oncogene-induced STAT3-activation is central to tumor progression by promoting cancer cell expression of pro-angiogenic and immunosuppressive factors. STAT3 is also activated in infiltrating immune cells including tumor-associated macrophages (TAM) amplifying immune suppression. Consequently, STAT3 is considered as a target for cancer therapy. However, its interplay with other STAT-family members or transcription factors such as NF-κB has to be considered in light of their concerted regulation of immune-related genes. Here, we discuss new attempts at re-educating immune suppressive tumor-associated macrophages towards a CD8 T cell supporting profile, with an emphasis on the role of STAT transcription factors on TAM functional programs. Recent clinical trials using JAK/STAT inhibitors highlighted the negative effects of these molecules on the maintenance and function of effector/memory T cells. Concerted regulation of STAT3 and STAT5 activation in CD8 T effector and memory cells has been shown to impact their tumor-specific responses including intra-tumor accumulation, long-term survival, cytotoxic activity and resistance toward tumor-derived immune suppression. Interestingly, as an escape mechanism, melanoma cells were reported to impede STAT5 nuclear translocation in both CD8 T cells and NK cells. Ours and others results will be discussed in the perspective of new developments in engineered T cell-based adoptive therapies to treat cancer patients.

The three members of the Vav family proteins form complexes that concur to foam cell formation and atherosclerosis.

During foam cell formation and atherosclerosis development, the scavenger receptor CD36 plays critical roles in lipid uptake and triggering of atherogenicity via the activation of Vav molecules. The Vav family includes three highly conserved members known as Vav1, Vav2, and Vav3. As Vav1 and Vav3 were found to exert function in atherosclerosis development, it remains thus to decipher whether Vav2 also plays a role in the development of atherosclerosis. In this study we found that Vav2 deficiency in RAW264.7 macrophages significantly diminished oxidized LDL uptake and CD36 signaling, demonstrating that each Vav protein family member was required for foam cell formation. Genetic disruption of Vav2 in ApoE-deficient C57BL/6 mice significantly inhibited the severity of atherosclerosis. Strikingly, we further found that the genetic deletion of each member of the Vav protein family by CRISPR/Cas9 resulted in a similar alteration of transcriptomic profiles of macrophages. The three members of the Vav proteins were found to form complexes, and genetic ablation of each single Vav molecule was sufficient to prevent endocytosis of CD36. The functional interdependence of the three Vav family members in foam cell formation was due to their indispensable roles in transcriptomic programing, lipid uptake, and activation of the JNK kinase in macrophages.

Tracking Dynamics of Spontaneous Tumors in Mice Using Photon-Counting Computed Tomography.

Computed tomography is a powerful medical imaging modality for longitudinal studies in cancer to follow neoplasia progression and evaluate anticancer therapies. Here, we report the generation of a photon-counting micro-computed tomography (PC-CT) method based on hybrid pixel detectors with enhanced sensitivity and precision of tumor imaging. We then applied PC-CT for longitudinal imaging in a clinically relevant liver cancer model, the Alb-R26Met mice, and found a remarkable heterogeneity in the dynamics for tumors at the initiation phases. Instead, the growth curve of evolving tumors exhibited a comparable exponential growth, with a constant doubling time. Furthermore, longitudinal PC-CT imaging in mice treated with a combination of MEK and BCL-XL inhibitors revealed a drastic tumor regression accompanied by a striking remodeling of macrophages in the tumor microenvironment. Thus, PC-CT is a powerful system to detect cancer initiation and progression, and to monitor its evolution during treatment.

Specific targeting of CD163+ TAMs mobilizes inflammatory monocytes and promotes T cell-mediated tumor regression.

Tumor-associated macrophages (TAMs) play critical roles in tumor progression but are also capable of contributing to antitumor immunity. Recent studies have revealed an unprecedented heterogeneity among TAMs in both human cancer and experimental models. Nevertheless, we still understand little about the contribution of different TAM subsets to tumor progression. Here, we demonstrate that CD163-expressing TAMs specifically maintain immune suppression in an experimental model of melanoma that is resistant to anti-PD-1 checkpoint therapy. Specific depletion of the CD163+ macrophages results in a massive infiltration of activated T cells and tumor regression. Importantly, the infiltration of cytotoxic T cells was accompanied by the mobilization of inflammatory monocytes that significantly contributed to tumor regression. Thus, the specific targeting of CD163+ TAMs reeducates the tumor immune microenvironment and promotes both myeloid and T cell-mediated antitumor immunity, illustrating the importance of selective targeting of tumor-associated myeloid cells in a therapeutic context.

Membrane Cholesterol Efflux Drives Tumor-Associated Macrophage Reprogramming and Tumor Progression.

Macrophages possess intrinsic tumoricidal activity, yet tumor-associated macrophages (TAMs) rapidly adopt an alternative phenotype within the tumor microenvironment that is marked by tumor-promoting immunosuppressive and trophic functions. The mechanisms that promote such TAM polarization remain poorly understood, but once identified, they may represent important therapeutic targets to block the tumor-promoting functions of TAMs and restore their anti-tumor potential. Here, we have characterized TAMs in a mouse model of metastatic ovarian cancer. We show that ovarian cancer cells promote membrane-cholesterol efflux and depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4-mediated reprogramming, including inhibition of IFNγ-induced gene expression. Genetic deletion of ABC transporters, which mediate cholesterol efflux, reverts the tumor-promoting functions of TAMs and reduces tumor progression. These studies reveal an unexpected role for membrane-cholesterol efflux in driving TAM-mediated tumor progression while pointing to a potentially novel anti-tumor therapeutic strategy.

Precise and Rapid Validation of Candidate Gene by Allele Specific Knockout With CRISPR/Cas9 in Wild Mice.

It is a tempting goal to identify causative genes underlying phenotypic differences among inbred strains of mice, which is a huge reservoir of genetic resources to understand mammalian pathophysiology. In particular, the wild-derived mouse strains harbor enormous genetic variations that have been acquired during evolutionary divergence over 100s of 1000s of years. However, validating the genetic variation in non-classical strains was extremely difficult, until the advent of CRISPR/Cas9 genome editing tools. In this study, we first describe a T cell phenotype in both wild-derived PWD/PhJ parental mice and F1 hybrids, from a cross to C57BL/6 (B6) mice, and we isolate a genetic locus on Chr2, using linkage mapping and chromosome substitution mice. Importantly, we validate the identification of the functional gene controlling this T cell phenotype, Cd44, by allele specific knockout of the PWD copy, leaving the B6 copy completely intact. Our experiments using F1 mice with a dominant phenotype, allowed rapid validation of candidate genes by designing sgRNA PAM sequences that only target the DNA of the PWD genome. We obtained 10 animals derived from B6 eggs fertilized with PWD sperm cells which were subjected to microinjection of CRISPR/Cas9 gene targeting machinery. In the newborns of F1 hybrids, 80% (n = 10) had allele specific knockout of the candidate gene Cd44 of PWD origin, and no mice showed mistargeting of the B6 copy. In the resultant allele-specific knockout F1 mice, we observe full recovery of T cell phenotype. Therefore, our study provided a precise and rapid approach to functionally validate genes that could facilitate gene discovery in classic mouse genetics. More importantly, as we succeeded in genetic manipulation of mice, allele specific knockout could provide the possibility to inactivate disease alleles while keeping the normal allele of the gene intact in human cells.