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2.
Cell Rep ; 42(12): 113507, 2023 12 26.
Article in English | MEDLINE | ID: mdl-38041815

ABSTRACT

The expression of pro-lymphangiogenic VEGF-C in primary tumors is associated with sentinel lymph node metastasis in most solid cancer types. However, the impact of VEGF-C on distant organ metastasis remains unclear. Perivascular tumor-associated macrophages (TAMs) play a crucial role in guiding hematogenous spread of cancer cells by establishing metastatic pathways within the tumor microenvironment. This process supports breast cancer cell intravasation and metastatic dissemination. We show here that VEGF-C-expressing TAMs reduce the dissemination of mammary cancer cells to the lungs while concurrently increasing lymph node metastasis. These TAMs express podoplanin and interact with normalized tumor blood vessels expressing VEGFR3. Moreover, clinical data suggest inverse association between VEGF-C-expressing TAMs and breast cancer malignancy. Thus, our study elucidates the paradoxical role of VEGF-C-expressing TAMs in redirecting cancer cells to preferentially disseminate to lymph nodes rather than to lungs, partially achieved by normalizing tumor blood vessels and promoting lymphangiogenesis.


Subject(s)
Breast Neoplasms , Humans , Female , Lymphatic Metastasis , Breast Neoplasms/pathology , Tumor-Associated Macrophages/metabolism , Vascular Endothelial Growth Factor C/metabolism , Lymphangiogenesis , Tumor Microenvironment
3.
Cancer Cell ; 41(11): 1892-1910.e10, 2023 11 13.
Article in English | MEDLINE | ID: mdl-37863068

ABSTRACT

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.


Subject(s)
CD8-Positive T-Lymphocytes , Liver Neoplasms , Humans , Mice , Animals , CTLA-4 Antigen/metabolism , Tumor Microenvironment/genetics , Macrophages , Liver Neoplasms/genetics , Liver Neoplasms/therapy , Liver Neoplasms/pathology
4.
JCI Insight ; 8(24)2023 Dec 22.
Article in English | MEDLINE | ID: mdl-37847559

ABSTRACT

Use of autologous cells isolated from elderly patients with multiple comorbidities may account for the modest efficacy of cell therapy in patients with chronic limb threatening ischemia (CLTI). We aimed to determine whether proarteriogenic monocyte/macrophages (Mo/MΦs) from patients with CLTI were functionally impaired and to demonstrate the mechanisms related to any impairment. Proarteriogenic Mo/MΦs isolated from patients with CLTI were found to have an impaired capacity to promote neovascularization in vitro and in vivo compared with those isolated from healthy controls. This was associated with increased expression of human HIV-1 TAT interactive protein-2 (HTATIP2), a transcription factor known to suppress angiogenesis/arteriogenesis. Silencing HTATIP2 restored the functional capacity of CLTI Mo/MΦs, which was associated with increased expression of arteriogenic regulators Neuropilin-1 and Angiopoietin-1, and their ability to enhance angiogenic (endothelial tubule formation) and arteriogenic (smooth muscle proliferation) processes in vitro. In support of the translational relevance of our findings, silencing HTATIP2 in proarteriogenic Mo/MΦs isolated from patients with CLTI rescued their capacity to enhance limb perfusion in the ischemic hindlimb by effecting greater angiogenesis and arteriogenesis. Ex vivo modulation of HTATIP2 may offer a strategy for rescuing the functional impairment of pro-angio/arteriogenic Mo/MΦs prior to autologous delivery and increase the likelihood of clinical efficacy.


Subject(s)
Monocytes , Neovascularization, Physiologic , Animals , Mice , Humans , Aged , Monocytes/metabolism , Collateral Circulation , Muscle, Skeletal/metabolism , Mice, Knockout , Ischemia/metabolism , Transcription Factors , Acetyltransferases
5.
Sci Immunol ; 7(74): eabn6563, 2022 08 12.
Article in English | MEDLINE | ID: mdl-35984893

ABSTRACT

Adoptive immunotherapy with T cells engineered with tumor-specific T cell receptors (TCRs) holds promise for cancer treatment. However, suppressive cues generated in the tumor microenvironment (TME) can hinder the efficacy of these therapies, prompting the search for strategies to overcome these detrimental conditions and improve cellular therapeutic approaches. CD1d-restricted invariant natural killer T (iNKT) cells actively participate in tumor immunosurveillance by restricting suppressive myeloid populations in the TME. Here, we showed that harnessing iNKT cells with a second TCR specific for a tumor-associated peptide generated bispecific effectors for CD1d- and major histocompatibility complex (MHC)-restricted antigens in vitro. Upon in vivo transfer, TCR-engineered iNKT (TCR-iNKT) cells showed the highest efficacy in restraining the progression of multiple tumors that expressed the cognate antigen compared with nontransduced iNKT cells or CD8+ T cells engineered with the same TCR. TCR-iNKT cells achieved robust cancer control by simultaneously modulating intratumoral suppressive myeloid populations and killing malignant cells. This dual antitumor function was further enhanced when the iNKT cell agonist α-galactosyl ceramide (α-GalCer) was administered as a therapeutic booster through a platform that ensured controlled delivery at the tumor site, named multistage vector (MSV). These preclinical results support the combination of tumor-redirected TCR-iNKT cells and local α-GalCer boosting as a potential therapy for patients with cancer.


Subject(s)
Natural Killer T-Cells , Neoplasms , Receptors, Antigen, T-Cell , Animals , Humans , Mice , CD8-Positive T-Lymphocytes , Cell Engineering , Myeloid Cells , Natural Killer T-Cells/physiology , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/therapeutic use , Tumor Microenvironment
6.
Nat Cardiovasc Res ; 1(5): 476-490, 2022 May.
Article in English | MEDLINE | ID: mdl-35602406

ABSTRACT

Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. VEGFA is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells towards progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs.

7.
Mol Ther Methods Clin Dev ; 19: 411-425, 2020 Dec 11.
Article in English | MEDLINE | ID: mdl-33294490

ABSTRACT

Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 109 transducing unit (TU)/mL and 5 × 104 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo, as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing.

8.
Proc Natl Acad Sci U S A ; 117(44): 27556-27565, 2020 11 03.
Article in English | MEDLINE | ID: mdl-33077599

ABSTRACT

Tumor-associated macrophages (TAMs) continuously fine tune their immune modulatory properties, but how gene expression programs coordinate this immune cell plasticity is largely unknown. Selective mRNA translation, controlled by MNK1/MNK2 and mTOR pathways impinging on eIF4E, facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs. Using polysome profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies. These alterations in gene expression paralleled accumulation of antiinflammatory macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, known to selectively modulate mRNA translation. Furthermore, suppression of the MNK2, but not the mTOR signaling pathway, reprogrammed antiinflammatory macrophages toward a proinflammatory phenotype with the ability to activate CD8+ T cells. Thus, selective changes of mRNA translation depending on MNK2 signaling represents a key node regulating macrophage antiinflammatory functions.


Subject(s)
Macrophages/immunology , Neoplasms/immunology , Protein Serine-Threonine Kinases/metabolism , Animals , Coculture Techniques , Disease Models, Animal , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Female , Gene Expression Regulation, Neoplastic/immunology , Gene Knockdown Techniques , Humans , MCF-7 Cells , Macrophages/metabolism , Mice , Mice, Transgenic , Naphthyridines/pharmacology , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation/genetics , Phosphorylation/immunology , Primary Cell Culture , Protein Serine-Threonine Kinases/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Tumor Escape/genetics
9.
J Clin Invest ; 130(3): 1199-1216, 2020 03 02.
Article in English | MEDLINE | ID: mdl-32015230

ABSTRACT

Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Colorectal Neoplasms , Drug Resistance, Neoplasm/drug effects , Immunotherapy , Neovascularization, Pathologic , Adenomatous Polyposis Coli Protein/genetics , Adenomatous Polyposis Coli Protein/immunology , Angiopoietin-2/genetics , Angiopoietin-2/immunology , Animals , Cell Line , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms/therapy , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/immunology , Homeodomain Proteins/genetics , Homeodomain Proteins/immunology , Humans , Matrix Metalloproteinase 14/genetics , Matrix Metalloproteinase 14/immunology , Mice , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/immunology , Neoplasms, Experimental/pathology , Neoplasms, Experimental/therapy , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/therapy , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/immunology , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/immunology
10.
Cell Rep ; 27(7): 1967-1978.e4, 2019 05 14.
Article in English | MEDLINE | ID: mdl-31091437

ABSTRACT

Lymphatic vasculature is crucial for metastasis in triple-negative breast cancer (TNBC); however, cellular and molecular drivers controlling lymphovascular metastasis are poorly understood. We define a macrophage-dependent signaling cascade that facilitates metastasis through lymphovascular remodeling. TNBC cells instigate mRNA changes in macrophages, resulting in ß4 integrin-dependent adhesion to the lymphovasculature. ß4 integrin retains macrophages proximal to lymphatic endothelial cells (LECs), where release of TGF-ß1 drives LEC contraction via RhoA activation. Macrophages promote gross architectural changes to lymphovasculature by increasing dilation, hyperpermeability, and disorganization. TGF-ß1 drives ß4 integrin clustering at the macrophage plasma membrane, further promoting macrophage adhesion and demonstrating the dual functionality of TGF-ß1 signaling in this context. ß4 integrin-expressing macrophages were identified in human breast tumors, and a combination of vascular-remodeling macrophage gene signature and TGF-ß signaling scores correlates with metastasis. We postulate that future clinical strategies for patients with TNBC should target crosstalk between ß4 integrin and TGF-ß1.


Subject(s)
Integrin beta4/metabolism , Lymphatic Vessels/cytology , Lymphatic Vessels/pathology , Macrophages/metabolism , Transforming Growth Factor beta1/metabolism , Triple Negative Breast Neoplasms/metabolism , Animals , Cell Adhesion/genetics , Cell Adhesion Molecules/metabolism , Endothelial Cells/metabolism , Female , Gene Expression Regulation, Neoplastic/genetics , HEK293 Cells , Humans , Integrin beta4/genetics , Lymphatic Metastasis , Lymphatic Vessels/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Signal Transduction/genetics , Transforming Growth Factor beta1/genetics , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism , Kalinin
11.
Hum Gene Ther ; 30(7): 893-905, 2019 07.
Article in English | MEDLINE | ID: mdl-30786776

ABSTRACT

Microvascular dysfunction and resulting tissue hypoxia is a major contributor to the pathogenesis and evolution of cardiovascular diseases (CVD). Diverse gene and cell therapies have been proposed to preserve the microvasculature or boost angiogenesis in CVD, with moderate benefit. This study tested in vivo the impact of sequential delivery by bone-marrow (BM) cells of the pro-angiogenic factors vascular endothelial growth factor (VEGFA) and sphingosine-1-phosphate (S1P) in a myocardial infarction model. For that, mouse BM cells were transduced with lentiviral vectors coding for VEGFA or sphingosine kinase (SPHK1), which catalyzes S1P production, and injected them intravenously 4 and 7 days after cardiac ischemia-reperfusion in mice. Sequential delivery by transduced BM cells of VEGFA and S1P led to increased endothelial cell numbers and shorter extravascular distances in the infarct zone, which support better oxygen diffusion 28 days post myocardial infarction, as shown by automated 3D image analysis of the microvasculature. Milder effects were observed in the remote zone, together with increased proportion of capillaries. BM cells delivering VEGFA and S1P also decreased myofibroblast abundance and restricted adverse cardiac remodeling without major impact on cardiac contractility. The results indicate that BM cells engineered to deliver VEGFA/S1P angiogenic factors sequentially may constitute a promising strategy to improve micro-vascularization and oxygen diffusion, thus limiting the adverse consequences of cardiac ischemia.


Subject(s)
Bone Marrow Cells/metabolism , Lysophospholipids/administration & dosage , Myocardial Infarction/genetics , Myocardial Infarction/therapy , Neovascularization, Pathologic/genetics , Sphingosine/analogs & derivatives , Vascular Endothelial Growth Factor A/genetics , Ventricular Remodeling/genetics , Animals , Biomarkers , Cell- and Tissue-Based Therapy , Disease Models, Animal , Genetic Therapy , Humans , Mice , Myocardial Infarction/diagnosis , Neovascularization, Pathologic/drug therapy , Sphingosine/administration & dosage , Ventricular Remodeling/drug effects
12.
Nat Cell Biol ; 21(2): 190-202, 2019 02.
Article in English | MEDLINE | ID: mdl-30598531

ABSTRACT

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.


Subject(s)
Doxorubicin/therapeutic use , Extracellular Vesicles/drug effects , Lung Neoplasms/drug therapy , Mammary Neoplasms, Experimental/drug therapy , Paclitaxel/therapeutic use , Animals , Annexin A6/metabolism , Cell Line, Tumor , Chemokine CCL2/metabolism , Extracellular Vesicles/metabolism , Female , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Mice, Transgenic
13.
Nat Methods ; 15(3): 183-186, 2018 03.
Article in English | MEDLINE | ID: mdl-29355847

ABSTRACT

We describe a lentivirus-encoded chimeric receptor, termed extracellular vesicle (EV)-internalizing receptor (EVIR), which enables the selective uptake of cancer-cell-derived EVs by dendritic cells (DCs). The EVIR enhances DC presentation of EV-associated tumor antigens to CD8+ T cells primarily through MHCI recycling and cross-dressing. EVIRs should facilitate exploring the mechanisms and implications of horizontal transfer of tumor antigens to antigen-presenting cells.


Subject(s)
Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Colonic Neoplasms/immunology , Dendritic Cells/immunology , Extracellular Vesicles/metabolism , Receptors, Chimeric Antigen/metabolism , Animals , Cells, Cultured , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Extracellular Vesicles/immunology , Female , Histocompatibility Antigens Class II/immunology , Mice , Mice, Inbred C57BL
14.
J Allergy Clin Immunol ; 141(1): 350-364.e8, 2018 01.
Article in English | MEDLINE | ID: mdl-28629744

ABSTRACT

BACKGROUND: Mannose receptor (MRC1/CD206) has been suggested to mediate allergic sensitization and asthma to multiple glycoallergens, including cockroach allergens. OBJECTIVE: We sought to determine the existence of a protective mechanism through which MRC1 limits allergic inflammation through its intronic miR-511-3p. METHODS: We examined MRC1-mediated cockroach allergen uptake by lung macrophages and lung inflammation using C57BL/6 wild-type (WT) and Mrc1-/- mice. The role of miR-511-3p in macrophage polarization and cockroach allergen-induced lung inflammation in mice transfected with adeno-associated virus (AAV)-miR-511-3p (AAV-cytomegalovirus-miR-511-3p-enhanced green fluorescent protein) was analyzed. Gene profiling of macrophages with or without miR-511-3p overexpression was also performed. RESULTS: Mrc1-/- lung macrophages showed a significant reduction in cockroach allergen uptake compared with WT mice, and Mrc1-/- mice had an exacerbated lung inflammation with increased levels of cockroach allergen-specific IgE and TH2/TH17 cytokines in a cockroach allergen-induced mouse model compared with WT mice. Macrophages from Mrc1-/- mice showed significantly reduced levels of miR-511-3 and an M1 phenotype, whereas overexpression of miR-511-3p rendered macrophages to exhibit a M2 phenotype. Furthermore, mice transfected with AAV-miR-511-3p showed a significant reduction in cockroach allergen-induced inflammation. Profiling of macrophages with or without miR-511-3p overexpression identified 729 differentially expressed genes, wherein expression of prostaglandin D2 synthase (Ptgds) and its product PGD2 were significantly downregulated by miR-511-3p. Ptgds showed a robust binding to miR-511-3p, which might contribute to the protective effect of miR-511-3p. Plasma levels of miR-511-3p were significantly lower in human asthmatic patients compared with nonasthmatic subjects. CONCLUSION: These studies support a critical but previously unrecognized role of MRC1 and miR-511-3p in protection against allergen-induced lung inflammation.


Subject(s)
Hypersensitivity/etiology , Hypersensitivity/metabolism , Lectins, C-Type/metabolism , Macrophage Activation/genetics , Macrophage Activation/immunology , Macrophages/immunology , Macrophages/metabolism , Mannose-Binding Lectins/metabolism , MicroRNAs/genetics , Receptors, Cell Surface/metabolism , Allergens/immunology , Animals , Asthma/etiology , Asthma/metabolism , Asthma/pathology , Cockroaches/immunology , Gene Expression Profiling , Gene Expression Regulation , Genetic Vectors/genetics , Hypersensitivity/pathology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/metabolism , Mannose Receptor , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Knockout , Models, Biological , Pneumonia/etiology , Pneumonia/metabolism , Pneumonia/pathology , RNA Interference , Receptors, Cell Surface/genetics , Receptors, Immunologic
15.
Sci Rep ; 7(1): 14655, 2017 11 07.
Article in English | MEDLINE | ID: mdl-29116108

ABSTRACT

Tumor-associated macrophages (TAMs) expressing the multi-ligand endocytic receptor mannose receptor (CD206/MRC1) contribute to tumor immunosuppression, angiogenesis, metastasis, and relapse. Here, we describe a peptide that selectively targets MRC1-expressing TAMs (MEMs). We performed in vivo peptide phage display screens in mice bearing 4T1 metastatic breast tumors to identify peptides that target peritoneal macrophages. Deep sequencing of the peptide-encoding inserts in the selected phage pool revealed enrichment of the peptide CSPGAKVRC (codenamed "UNO"). Intravenously injected FAM-labeled UNO (FAM-UNO) homed to tumor and sentinel lymph node MEMs in different cancer models: 4T1 and MCF-7 breast carcinoma, B16F10 melanoma, WT-GBM glioma and MKN45-P gastric carcinoma. Fluorescence anisotropy assay showed that FAM-UNO interacts with recombinant CD206 when subjected to reducing conditions. Interestingly, the GSPGAK motif is present in all CD206-binding collagens. FAM-UNO was able to transport drug-loaded nanoparticles into MEMs, whereas particles without the peptide were not taken up by MEMs. In ex vivo organ imaging, FAM-UNO showed significantly higher accumulation in sentinel lymph nodes than a control peptide. This study suggests applications for UNO peptide in diagnostic imaging and therapeutic targeting of MEMs in solid tumors.


Subject(s)
Drug Delivery Systems , Lectins, C-Type/metabolism , Macrophages/drug effects , Mammary Neoplasms, Experimental/drug therapy , Mannose-Binding Lectins/metabolism , Peptides/therapeutic use , Receptors, Cell Surface/metabolism , Animals , Drug Delivery Systems/methods , Female , Mannose Receptor , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Recombinant Proteins
16.
Arterioscler Thromb Vasc Biol ; 36(11): 2203-2212, 2016 11.
Article in English | MEDLINE | ID: mdl-27634833

ABSTRACT

OBJECTIVE: Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions. APPROACH AND RESULTS: We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents. CONCLUSIONS: This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.


Subject(s)
Capillaries/metabolism , Capillary Permeability , Cell Communication , Endothelial Cells/metabolism , Macrophages, Peritoneal/metabolism , Mesentery/blood supply , Peritoneum/blood supply , Skin/blood supply , Animals , Antigens, CD/metabolism , Cadherins/metabolism , Cell Movement , Cells, Cultured , Coculture Techniques , Dextrans/metabolism , Fluorescein-5-isothiocyanate/metabolism , Humans , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Ovalbumin/metabolism , Phenotype , Phosphorylation , Rhodamines/metabolism , Time Factors , Transfection
17.
Cell Cycle ; 15(23): 3149-3150, 2016 12.
Article in English | MEDLINE | ID: mdl-27494148
18.
Nat Cell Biol ; 18(7): 790-802, 2016 07.
Article in English | MEDLINE | ID: mdl-27295554

ABSTRACT

Tumour-associated macrophages (TAMs) largely express an alternatively activated (or M2) phenotype, which entails immunosuppressive and tumour-promoting capabilities. Reprogramming TAMs towards a classically activated (M1) phenotype may thwart tumour-associated immunosuppression and unleash anti-tumour immunity. Here we show that conditional deletion of the microRNA (miRNA)-processing enzyme DICER in macrophages prompts M1-like TAM programming, characterized by hyperactive IFN-γ/STAT1 signalling. This rewiring abated the immunosuppressive capacity of TAMs and fostered the recruitment of activated cytotoxic T lymphocytes (CTLs) to the tumours. CTL-derived IFN-γ exacerbated M1 polarization of Dicer1-deficient TAMs and inhibited tumour growth. Remarkably, DICER deficiency in TAMs negated the anti-tumoral effects of macrophage depletion by anti-CSF1R antibodies, and enabled complete tumour eradication by PD1 checkpoint blockade or CD40 agonistic antibodies. Finally, genetic rescue of Let-7 miRNA activity in Dicer1-deficient TAMs partly restored their M2-like phenotype and decreased tumour-infiltrating CTLs. These findings suggest that DICER/Let-7 activity opposes IFN-γ-induced, immunostimulatory M1-like TAM activation, with potential therapeutic implications.


Subject(s)
DEAD-box RNA Helicases/metabolism , Interferon-gamma/metabolism , Macrophage Activation/immunology , Macrophages/immunology , MicroRNAs/genetics , Ribonuclease III/metabolism , Tumor Microenvironment/genetics , Animals , Cells, Cultured , DEAD-box RNA Helicases/deficiency , Humans , Mice , Neoplasms/genetics , Neoplasms/immunology , Ribonuclease III/deficiency
19.
Cancer Res ; 76(11): 3166-78, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27197153

ABSTRACT

Accumulation of tumor-associated macrophages (TAM) correlates with malignant progression, immune suppression, and poor prognosis. In this study, we defined a critical role for the cell-surface guidance molecule SEMA3A in differential proliferative control of TAMs. Tumor cell-derived SEMA3A restricted the proliferation of protumoral M2 macrophages but increased the proliferation of antitumoral M1, acting through the SEMA3A receptor neuropilin 1. Expansion of M1 macrophages in vivo enhanced the recruitment and activation of natural killer (NK) cells and cytotoxic CD8(+) T cells to tumors, inhibiting their growth. In human breast cancer specimens, we found that immunohistochemical levels of SEMA3A correlated with the expression of genes characteristic of M1 macrophages, CD8(+) T cells, and NK cells, while inversely correlating with established characters of malignancy. In summary, our results illuminate a mechanism whereby the TAM phenotype is controlled and identify the cell-surface molecule SEMA3A as a candidate for therapeutic targeting. Cancer Res; 76(11); 3166-78. ©2016 AACR.


Subject(s)
Breast Neoplasms/pathology , CD8-Positive T-Lymphocytes/pathology , Cell Proliferation , Killer Cells, Natural/pathology , Macrophages/pathology , Semaphorin-3A/metabolism , Animals , Apoptosis , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , CD8-Positive T-Lymphocytes/metabolism , Disease Progression , Female , Humans , Immunoenzyme Techniques , Killer Cells, Natural/metabolism , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Nude , Neoplasm Grading , Neuropilin-1/genetics , Neuropilin-1/metabolism , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Semaphorin-3A/genetics , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
20.
Nat Commun ; 6: 8900, 2015 Nov 23.
Article in English | MEDLINE | ID: mdl-26592194

ABSTRACT

Despite its importance during viral or bacterial infections, transcriptional regulation of the interferon-ß gene (Ifnb1) in activated macrophages is only partially understood. Here we report that TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts. In macrophages, TRIM33 is recruited by PU.1 to a conserved region, the Ifnb1 Control Element (ICE), located 15 kb upstream of the Ifnb1 transcription start site. ICE constitutively interacts with Ifnb1 through a TRIM33-independent chromatin loop. At late phases of lipopolysaccharide activation of macrophages, TRIM33 is bound to ICE, regulates Ifnb1 enhanceosome loading, controls Ifnb1 chromatin structure and represses Ifnb1 gene transcription by preventing recruitment of CBP/p300. These results characterize a previously unknown mechanism of macrophage-specific regulation of Ifnb1 transcription whereby TRIM33 is critical for Ifnb1 gene transcription shutdown.


Subject(s)
Gene Expression Regulation , Interferon-beta/genetics , Macrophages/cytology , Macrophages/metabolism , Transcription Factors/metabolism , Animals , Female , Interferon-beta/metabolism , Macrophage Activation , Male , Mice , Mice, Inbred C57BL , Transcription Factors/genetics , Transcription, Genetic
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