ABSTRACT
Direct injection of therapies into tumors has emerged as an administration route capable of achieving high local drug exposure and strong anti-tumor response. A diverse array of immune agonists ranging in size and target are under development as local immunotherapies. However, due to the relatively recent adoption of intratumoral administration, the pharmacokinetics of locally-injected biologics remains poorly defined, limiting rational design of tumor-localized immunotherapies. Here we define a pharmacokinetic framework for biologics injected intratumorally that can predict tumor exposure and effectiveness. We find empirically and computationally that extending the tumor exposure of locally-injected interleukin-2 by increasing molecular size and/or improving matrix-targeting affinity improves therapeutic efficacy in mice. By tracking the distribution of intratumorally-injected proteins using positron emission tomography, we observe size-dependent enhancement in tumor exposure occurs by slowing the rate of diffusive escape from the tumor and by increasing partitioning to an apparent viscous region of the tumor. In elucidating how molecular weight and matrix binding interplay to determine tumor exposure, our model can aid in the design of intratumoral therapies to exert maximal therapeutic effect.
Subject(s)
Collagen/genetics , Immunotherapy/methods , Interleukin-2/pharmacology , Melanoma, Experimental/therapy , Receptors, Immunologic/genetics , Skin Neoplasms/therapy , Allografts , Animals , Carrier Proteins/genetics , Carrier Proteins/immunology , Cell Line, Tumor , Collagen/immunology , Female , Gene Library , Injections, Intralesional , Interleukin-2/genetics , Interleukin-2/immunology , Interleukin-2/pharmacokinetics , Melanoma, Experimental/diagnostic imaging , Melanoma, Experimental/genetics , Melanoma, Experimental/mortality , Mice , Mice, Inbred C57BL , Peptides/genetics , Peptides/immunology , Positron-Emission Tomography , Protein Binding , Protein Engineering/methods , Receptors, Immunologic/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Serum Albumin/genetics , Serum Albumin/immunology , Skin Neoplasms/diagnostic imaging , Skin Neoplasms/genetics , Skin Neoplasms/mortality , Survival Analysis , Tumor Burden/drug effectsABSTRACT
For many solid tumors, immune checkpoint blockade therapy has become first line treatment, yet a large proportion of patients with immunologically cold tumors do not benefit due to the paucity of tumor infiltrating lymphocytes. Here we show that the orphan G Protein-Coupled Receptor 182 (GPR182) contributes to immunotherapy resistance in cancer via scavenging chemokines that are important for lymphocyte recruitment to tumors. GPR182 is primarily upregulated in melanoma-associated lymphatic endothelial cells (LECs) during tumorigenesis, and this atypical chemokine receptor endocytoses chemokines promiscuously. In GPR182-deficient mice, T cell infiltration into transplanted melanomas increases, leading to enhanced effector T cell function and improved antitumor immunity. Ablation of GPR182 leads to increased intratumoral concentrations of multiple chemokines and thereby sensitizes poorly immunogenic tumors to immune checkpoint blockade and adoptive cellular therapies. CXCR3 blockade reverses the improved antitumor immunity and T cell infiltration characteristic of GPR182-deficient mice. Our study thus identifies GPR182 as an upstream regulator of the CXCL9/CXCL10/CXCR3 axis that limits antitumor immunity and as a potential therapeutic target in immunologically cold tumors.
Subject(s)
Chemokine CXCL10/genetics , Chemokine CXCL9/genetics , Melanoma, Experimental/genetics , Melanoma/genetics , Receptors, CXCR3/genetics , Receptors, G-Protein-Coupled/genetics , Skin Neoplasms/genetics , Animals , Cell Movement , Chemokine CXCL10/immunology , Chemokine CXCL9/immunology , Gene Expression Regulation, Neoplastic , Humans , Immunotherapy/methods , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/immunology , Melanoma/mortality , Melanoma/therapy , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/therapy , Mice , Mice, Knockout , Protein Binding , Receptors, CXCR3/immunology , Receptors, G-Protein-Coupled/immunology , Signal Transduction , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Skin Neoplasms/therapy , Survival Analysis , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantation , Tumor Burden , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Microbe-based cancer immunotherapy has recently emerged as a hot topic for cancer treatment. However, serious limitations remain including infection associated side-effect and unsatisfactory outcomes in clinic trials. Here, we fabricate different sizes of nano-formulations derived from yeast cell wall (YCW NPs) by differential centrifugation. The induction of anticancer immunity of our formulations appears to inversely correlate with their size due to the ability to accumulate in tumor-draining lymph node (TDLN). Moreover, we use a percolation model to explain their distribution behavior toward TDLN. The abundance and functional orientation of each effector component are significantly improved not only in the microenvironment in tumor but also in the TDLN following small size YCW NPs treatment. In combination with programmed death-ligand 1 (PD-L1) blockade, we demonstrate anticancer efficiency in melanoma-challenged mice. We delineate potential strategy to target immunosuppressive microenvironment by microbe-based nanoparticles and highlight the role of size effect in microbe-based immune therapeutics.
Subject(s)
Immunotherapy/methods , Lymph Nodes/drug effects , Melanoma, Experimental/therapy , Nanoparticles/administration & dosage , Saccharomyces cerevisiae/chemistry , Skin Neoplasms/therapy , Allografts , Animals , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/genetics , B7-H1 Antigen/immunology , Cell Line, Tumor , Cell Wall/chemistry , Dendritic Cells/drug effects , Dendritic Cells/immunology , Female , Gene Expression Regulation, Neoplastic , Injections, Intralesional , Lymph Nodes/immunology , Lymph Nodes/pathology , Macrophage Activation/drug effects , Melanoma, Experimental/genetics , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Nanoparticles/chemistry , Particle Size , RAW 264.7 Cells/drug effects , RAW 264.7 Cells/immunology , Skin Neoplasms/genetics , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Survival Analysis , Syk Kinase/antagonists & inhibitors , Syk Kinase/genetics , Syk Kinase/immunology , Toll-Like Receptor 2/antagonists & inhibitors , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/immunology , Tumor Burden/drug effects , Tumor Microenvironment/drug effectsABSTRACT
BACKGROUND: Surgical resection remains an important component of multimodality treatment for most solid tumors. Neoadjuvant immunotherapy has several potential advantages, including in-situ tumor vaccination and pathologic assessment of response in the surgical specimen. We previously described an in-situ tumor vaccination strategy in melanoma using local radiation (RT) and an intratumoral injection of tumor-specific anti-GD2 immunocytokine (IT-IC). Here we tested whether neoadjuvant in-situ tumor vaccination using anti-GD2 immunocytokine and surgical resection, without RT, could generate immunologic memory capable of preventing recurrence or distant metastasis. METHODS: Mice bearing GD2 expressing B78 melanoma tumors were treated with neoadjuvant radiation, IT-IC, or combined RT + IT-IC. Surgical resection was performed following neoadjuvant immunotherapy. Immune infiltrate was assessed in the resection specimens. Mice were rechallenged with either B78 contralateral flank tumors or pulmonary seeding of non-GD2 expressing B16 melanoma metastasis induced experimentally. Rejection of rechallenge in mice treated with the various treatment regimens was considered evidence of immunologic memory. RESULTS: Neoadjuvant IT-IC and surgical resection resulted in increased CD8 T cell infiltration, a higher CD8:regulatory T cell ratio, and immunologic memory against contralateral flank rechallenge. The timing of resection did not significantly impact the development of memory, which was present as early as the day of surgery. There was less local wound toxicity with neoadjuvant IT-IC compared with neoadjuvant RT +IT IC. Neoadjuvant IT-IC and resection resulted in the rejection of B16 lung metastasis in a CD4 T cell dependent manner. CONCLUSIONS: Neoadjuvant IT-IC generates immunologic memory capable of preventing distant metastasis despite limited efficacy against large primary melanoma tumors. By combining neoadjuvant tumor vaccination and surgery, the toxicity of local RT was avoided. These preclinical data support further investigation regarding the use of neoadjuvant IT-IC in patients with melanoma at high risk for occult distant disease.
Subject(s)
Melanoma, Experimental , Neoadjuvant Therapy , Vaccination , Animals , Female , Mice , Disease Models, Animal , Melanoma, Experimental/drug therapy , Melanoma, Experimental/mortality , Neoadjuvant Therapy/methods , Survival Analysis , Vaccination/methodsABSTRACT
Adoptive T cell therapy (ACT) requires lymphodepletion preconditioning to eliminate immune-suppressive elements and enable efficient engraftment of adoptively transferred tumor-reactive T cells. As anti-CD4 monoclonal antibody depletes CD4+ immune-suppressive cells, the combination of anti-CD4 treatment and ACT has synergistic potential in cancer therapy. Here, we demonstrate a post-ACT conditioning regimen that involves transient anti-CD4 treatment (CD4post). Using murine melanoma, the combined effect of cyclophosphamide preconditioning (CTXpre), CD4post, and ex vivo primed tumor-reactive CD8+ T-cell infusion is presented. CTXpre/CD4post increases tumor suppression and host survival by accelerating the proliferation and differentiation of ex vivo primed CD8+ T cells and endogenous CD8+ T cells. Endogenous CD8+ T cells enhance effector profile and tumor-reactivity, indicating skewing of the TCR repertoire. Notably, enrichment of polyfunctional IL-18Rαhi CD8+ T cell subset is the key event in CTXpre/CD4post-induced tumor suppression. Mechanistically, the anti-tumor effect of IL-18Rαhi subset is mediated by IL-18 signaling and TCR-MHC I interaction. This study highlights the clinical relevance of CD4post in ACT and provides insights regarding the immunological nature of anti-CD4 treatment, which enhances anti-tumor response of CD8+ T cells.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Alkylating/pharmacology , CD4-Positive T-Lymphocytes/drug effects , Cyclophosphamide/pharmacology , Interleukin-18 Receptor alpha Subunit/genetics , Melanoma, Experimental/therapy , Skin Neoplasms/therapy , T-Lymphocytes, Cytotoxic/immunology , Adoptive Transfer , Animals , Antigens, CD/genetics , Antigens, CD/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Female , Gene Expression Regulation, Neoplastic , Immunotherapy, Adoptive/methods , Interleukin-18/genetics , Interleukin-18/immunology , Interleukin-18 Receptor alpha Subunit/agonists , Interleukin-18 Receptor alpha Subunit/immunology , Lymphocyte Activation , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Mice , Mice, Inbred C57BL , Receptors, CCR4/genetics , Receptors, CCR4/immunology , Receptors, CCR8/genetics , Receptors, CCR8/immunology , Receptors, Histamine H4/genetics , Receptors, Histamine H4/immunology , Signal Transduction , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Survival Analysis , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/transplantation , Tumor Burden/drug effectsABSTRACT
Prognosis and treatment of metastatic cancer continues to be one of the most difficult and challenging areas of oncology. Treatment usually consists of chemotherapeutics, which may be ineffective due to drug resistance, adverse effects, and dose-limiting toxicity. Therefore, novel approaches such as immunotherapy have been investigated to improve patient outcomes and minimize side effects. S100A9 is a calcium-binding protein implicated in tumor metastasis, progression, and aggressiveness that modulates the tumor microenvironment into an immunosuppressive state. S100A9 is expressed in and secreted by immune cells in the pre-metastatic niche, as well as, post-tumor development, therefore making it a suitable targeted for prophylaxis and therapy. In previous work, it is demonstrated that cowpea mosaic virus (CPMV) acts as an adjuvant when administered intratumorally. Here, it is demonstrated that systemically administered, S100A9-targeted CPMV homes to the lungs leading to recruitment of innate immune cells. This approach is efficacious both prophylactically and therapeutically against lung metastasis from melanoma and breast cancer. The current research will facilitate and accelerate the development of next-generation targeted immunotherapies administered as prophylaxis, that is, after surgery of a primary breast tumor to prevent outgrowth of metastasis, as well as, therapy to treat established metastatic disease.
Subject(s)
Breast Neoplasms/pathology , Calgranulin B/metabolism , Comovirus/immunology , Melanoma, Experimental/pathology , Nanoparticles/chemistry , Animals , Breast Neoplasms/mortality , Calgranulin B/chemistry , Cell Line, Tumor , Comovirus/chemistry , Female , Humans , Immunotherapy , Lung Neoplasms/secondary , Lung Neoplasms/therapy , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Melanoma, Experimental/mortality , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nanoparticles/metabolism , Nanoparticles/therapeutic use , Peptides/chemistry , Pre-Exposure Prophylaxis , Survival RateABSTRACT
Immunotherapy is revolutionizing cancer treatment. Vaccination of antigenic peptides has been identified as a promising strategy for cancer immunotherapy while insufficient immune responses were stimulated due to low antigenicity. Moreover, immune checkpoint blockade therapy is still limited by a low objective response rate. In this work, cationic polymer-lipid hybrid nanovesicle (P/LNV)-based liposomes are designed to simultaneously deliver tumor vaccines composed of anionic antigen epitopes, toll-like receptor-9 agonist (TLR9), CpG (AE/CpG), and indoleamine-2,3-dioxygenase (IDO) inhibitor, 1-methyl-tryptophan (1-MT), to increase the immunogenicity of peptide antigens and meanwhile block the immune checkpoint. P/LNV liposomes efficiently enhanced the uptake of vaccines by dendritic cells (DCs) and improved the maturation of DCs indicated by the significantly increased percentage of CD86+MHCI+ DCs, resulting in a potent cytotoxic T-lymphocyte (CTL) response against B16-OVA tumor cells in vitro. Importantly, the combination immunotherapy showed significantly higher therapeutic efficiency towards melanoma tumors in mice, compared with an untreated or individual therapy modality. Mechanistically, the co-delivery system could elicit a strong cancer-specific T-cell response, as characterized by the remarkably increased infiltration of CD8+ T cells in the tumor and draining lymph nodes. Altogether, cationic liposomes delivered with tumor vaccines and IDO inhibitor provide a promising platform for cancer immunotherapy by provoking antitumor T-cell immunity and simultaneously reversing the immunosuppressive tumor microenvironment.
Subject(s)
CpG Islands , Epitopes/immunology , Immunotherapy/methods , Liposomes/chemistry , Melanoma, Experimental/therapy , Tryptophan/analogs & derivatives , Animals , Anions/chemistry , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/administration & dosage , Cancer Vaccines/chemistry , Cancer Vaccines/immunology , Cations/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Epitopes/chemistry , Lipids/chemistry , Liposomes/pharmacology , Melanoma, Experimental/mortality , Mice , Mice, Inbred C57BL , Nanostructures/chemistry , Polymers/chemistry , Survival Rate , Tryptophan/chemistryABSTRACT
Metastasis is the main cause of death in cancer patients; therefore, new strategies or technologies that can inhibit the growth of primary tumors and their metastatic spread are extremely valuable. In this study, we selected an E-selectin-binding peptide as a targeting ligand and an inhibitor of metastasis, and conjugated this peptide with SN38 and PEG to produce an amphiphilic PEGylated peptide-drug conjugate (PDC). Novel self-assembled nanoparticles were then formed by the amphiphilic conjugate. The particles were actively targeted to the tumor vasculature by the peptide and passively to the tumor site by the enhanced permeability and retention (EPR) effect. As a nano-prodrug, this multifunctional conjugate (PEG-Pep-SN38) could reduce tumor growth, with an effect similar to that of irinotecan. Moreover, it could prolong the survival of mice bearing primary HCT116 tumors, which was not observed for its parent drug, SN38, nor the clinical prodrug of SN38 (irinotecan). Furthermore, this PDC prodrug prevented B16-F10 colonization in the lungs of mice. This study describes a new tumor vasculature-targeting PDC nano-prodrug with convenient preparation and high potential for cancer therapy, with the potential to be applied to other chemotherapeutic drugs.
Subject(s)
Irinotecan/chemistry , Nanoparticles/chemistry , Peptides/chemistry , Polyethylene Glycols/chemistry , Prodrugs/chemistry , Animals , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Humans , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Melanoma, Experimental/drug therapy , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Nude , Prodrugs/pharmacology , Prodrugs/therapeutic use , Survival Rate , Transplantation, HeterologousABSTRACT
The two T cell inhibitory receptors PD-1 and TIM-3 are co-expressed during exhausted T cell differentiation, and recent evidence suggests that their crosstalk regulates T cell exhaustion and immunotherapy efficacy; however, the molecular mechanism is unclear. Here we show that PD-1 contributes to the persistence of PD-1+TIM-3+ T cells by binding to the TIM-3 ligand galectin-9 (Gal-9) and attenuates Gal-9/TIM-3-induced cell death. Anti-Gal-9 therapy selectively expands intratumoral TIM-3+ cytotoxic CD8 T cells and immunosuppressive regulatory T cells (Treg cells). The combination of anti-Gal-9 and an agonistic antibody to the co-stimulatory receptor GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) that depletes Treg cells induces synergistic antitumor activity. Gal-9 expression and secretion are promoted by interferon ß and γ, and high Gal-9 expression correlates with poor prognosis in multiple human cancers. Our work uncovers a function for PD-1 in exhausted T cell survival and suggests Gal-9 as a promising target for immunotherapy.
Subject(s)
Adenocarcinoma/therapy , Colonic Neoplasms/therapy , Galectins/immunology , Gene Expression Regulation, Neoplastic/immunology , Glucocorticoid-Induced TNFR-Related Protein/immunology , Hepatitis A Virus Cellular Receptor 2/immunology , Programmed Cell Death 1 Receptor/immunology , Adenocarcinoma/genetics , Adenocarcinoma/immunology , Adenocarcinoma/mortality , Animals , Antibodies/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Colonic Neoplasms/genetics , Colonic Neoplasms/immunology , Colonic Neoplasms/mortality , Galectins/antagonists & inhibitors , Galectins/genetics , Glucocorticoid-Induced TNFR-Related Protein/agonists , Glucocorticoid-Induced TNFR-Related Protein/genetics , Hepatitis A Virus Cellular Receptor 2/genetics , Humans , Immunotherapy/methods , Jurkat Cells , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/therapy , Mice , Mice, Inbred BALB C , Programmed Cell Death 1 Receptor/genetics , Protein Binding , Signal Transduction , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Skin Neoplasms/therapy , Survival Analysis , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/pathology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathologyABSTRACT
A novel STING agonist, CDGSF, ipsilaterally modified with phosphorothioate and fluorine, was synthesized. The phosphorothioate in CDGSF might be a site for covalent conjugation. Injection of CDGSF generated an immunogenic ("hot") tumor microenvironment to suppress melanoma, more efficiently than dithio CDG. In particular, immunization with SARS-CoV-2 spike protein using CDGSF as an adjuvant elicited an exceptionally high antibody titer and a robust T cell response, overcoming the drawbacks of aluminum hydroxide. These results highlighted the therapeutic potential of CDGSF for cancer immunotherapy and the adjuvant potential of the STING agonist in the SARS-CoV-2 vaccine for the first time.
Subject(s)
Adjuvants, Immunologic/administration & dosage , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Melanoma, Experimental/drug therapy , Membrane Proteins/agonists , Nucleotides, Cyclic/administration & dosage , Skin Neoplasms/drug therapy , Adjuvants, Immunologic/chemical synthesis , Aluminum Hydroxide/administration & dosage , Aluminum Hydroxide/chemistry , Animals , Antibodies, Viral/biosynthesis , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/virology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/chemistry , Enzyme-Linked Immunospot Assay , Humans , Immunotherapy/methods , Interferon-gamma/biosynthesis , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Nucleotides, Cyclic/chemical synthesis , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Survival Analysis , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/virology , Tumor Burden/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Vaccination/methodsABSTRACT
Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.
Subject(s)
Immunotherapy/methods , Melanoma, Experimental/therapy , Neuraminidase/immunology , Polysaccharides/chemistry , Receptor, ErbB-2/chemistry , Sialic Acid Binding Immunoglobulin-like Lectins/immunology , Allografts , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , B7-H1 Antigen/genetics , B7-H1 Antigen/immunology , Cell Line, Tumor , Humans , Hydrolysis , Immunoconjugates/chemistry , Immunoconjugates/metabolism , Immunoconjugates/pharmacology , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Molecular Targeted Therapy , Neuraminidase/chemistry , Neuraminidase/genetics , Polysaccharides/immunology , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/immunology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptor, ErbB-2/genetics , Receptor, ErbB-2/immunology , Sialic Acid Binding Immunoglobulin-like Lectins/chemistry , Sialic Acid Binding Immunoglobulin-like Lectins/genetics , Survival Analysis , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
BACKGROUND: This study aimed to evaluate the relationship between survivin expression and melanoma after 5-aminolevulinic acid (5-ALA)-mediated sonodynamic therapy. METHODS: Immunohistochemistry was used to detect survivin protein expression in human melanoma clinical samples. Subsequently, the effects of 5-ALA-mediated sonodynamic therapy were determined by measuring the volume of melanoma xenografts and the bodyweights of melanoma-bearing nude mice. The MTT assay was used to detect the viability of melanoma B16-F10 cells under the action of 5-ALA-mediated sonodynamic therapy, and Western blotting and PCR were used to detect survivin expression in melanoma cells and in the melanoma-xenograft model. RESULTS: Survivin expression was significantly upregulated in human melanoma tissues compared with that of non-melanoma tissues. In the in vivo case, 5-ALA-mediated sonodynamic therapy significantly delayed tumor growth, prolonged the survival of mice, and inhibited the expression of survivin. In the in vitro case, 5-ALA-mediated sonodynamic therapy inhibited B16-F10 cell proliferation and decreased survivin expression at both protein and mRNA levels. CONCLUSION: Our results suggest that 5-ALA-mediated sonodynamic therapy inhibited B16-F10 cell proliferation and melanoma-xenograft growth and prolonged survival of melanoma-bearing nude mice, which might be through downregulation of survivin expression.
Subject(s)
Aminolevulinic Acid/administration & dosage , Melanoma, Experimental/therapy , Skin Neoplasms/therapy , Survivin/antagonists & inhibitors , Ultrasonic Therapy/methods , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Combined Modality Therapy/methods , Down-Regulation/drug effects , Down-Regulation/radiation effects , Humans , Male , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Prognosis , Skin/pathology , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Survival Analysis , Survivin/metabolism , Time Factors , Up-RegulationABSTRACT
BACKGROUND: The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. RESULTS: LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. CONCLUSIONS: The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.
Subject(s)
Angiogenesis Inhibitors/genetics , Genetic Therapy/methods , Liposomes/chemistry , Melanoma, Experimental/therapy , Receptors, Vascular Endothelial Growth Factor/metabolism , 3T3 Cells , Animals , Cell Line, Tumor , Cell Proliferation , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Male , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Plasmids/chemistry , Plasmids/genetics , Plasmids/therapeutic use , Protein Domains/genetics , Receptors, Vascular Endothelial Growth Factor/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Survival Rate , Transplantation, Homologous , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Experiments on F1(CBA×C57BL/6) mice with experimental metastatic melanoma B16 F10 showed that single intravenous injection of xenogeneic bone marrow mesenchymal stromal cells (BM-MSC) in a dose of 106 cells/mouse significantly increased 100-day survival rate of tumor-bearing animals. In contrast, administration of BM-MSC in a dose of 2×106 cells/ mouse reduced survival rates in comparison with the biocontrol (injection of B16 cells alone, 5×105 cells/mouse). This phenomenon can be related to in vivo participation of BM-MSC in reprogramming of resident tissue macrophages, including tumor microenvironment, towards pro- (M1) or anti-inflammatory (M2) phenotype. This is indirectly confirmed by the data on switching from activation to inhibition of ROS-producing activity of blood mononuclears and peritoneal macrophages in tumor-bearing mice in the test of luminol-dependent zymosaninduced chemiluminescence.
Subject(s)
Lung Neoplasms/therapy , Macrophages/immunology , Melanoma, Experimental/therapy , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/immunology , Skin Neoplasms/therapy , Administration, Intravenous , Animals , Cell Count , Cellular Reprogramming/genetics , Cellular Reprogramming/immunology , Female , Humans , Lung Neoplasms/immunology , Lung Neoplasms/mortality , Lung Neoplasms/secondary , Macrophages/pathology , Male , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/secondary , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Reactive Oxygen Species/immunology , Reactive Oxygen Species/metabolism , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Survival Analysis , Transplantation, Heterologous , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
The use of microneedles has facilitated the painless localized delivery of drugs across the skin. However, their efficacy has been limited by slow diffusion of molecules and often requires external triggers. Herein, an autonomous and degradable, active microneedle delivery platform is introduced, employing magnesium microparticles loaded within the microneedle patch, as the built-in engine for deeper and faster intradermal payload delivery. The magnesium particles react with the interstitial fluid, leading to an explosive-like rapid production of H2 bubbles, providing the necessary force to breach dermal barriers and enhance payload delivery. The release kinetics of active microneedles is evaluated in vitro by measuring the amount of IgG antibody (as a model drug) that passed through phantom tissue and a pigskin barrier. In vivo experiments using a B16F10 mouse melanoma model demonstrate that the active delivery of anti-CTLA-4 (a checkpoint inhibitor drug) results in greatly enhanced immune response and significantly longer survival. Moreover, spatially resolved zones of active and passive microneedles allow a combinatorial rapid burst response along with slow, sustained release, respectively. Such versatile and effective autonomous dynamic microneedle delivery technology offers considerable promise for a wide range of therapeutic applications, toward a greatly enhanced outcome, convenience, and cost.
Subject(s)
Drug Delivery Systems/methods , Needles , Administration, Cutaneous , Animals , Antibodies/immunology , Antibodies/therapeutic use , CTLA-4 Antigen/antagonists & inhibitors , CTLA-4 Antigen/metabolism , Humans , Immunotherapy , Melanoma, Experimental/drug therapy , Melanoma, Experimental/mortality , Mice, Inbred C57BL , MicroinjectionsABSTRACT
Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.
Subject(s)
CD47 Antigen/antagonists & inhibitors , CTLA-4 Antigen/antagonists & inhibitors , Ipilimumab/administration & dosage , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma, Experimental/mortality , T-Lymphocytes, Cytotoxic/immunology , Animals , CD47 Antigen/genetics , CD47 Antigen/immunology , Combined Modality Therapy , Humans , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/radiation effects , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Melanoma, Experimental/therapy , Mice , Mice, Inbred C57BL , Radiation Dosage , Survival Rate , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/radiation effects , Tumor Cells, CulturedABSTRACT
Regulatory T (Treg) cells are essential for peripheral tolerance and rely on the transcription factor (TF) Foxp3 for their generation and function. Several other TFs are critical for the Treg cell program. We found that mice deficient in Bcl11b TF solely in Treg cells developed fatal autoimmunity, and Bcl11b-deficient Treg cells had severely altered function. Bcl11b KO Treg cells showed decreased functional marker levels in homeostatic conditions, inflammation, and tumors. Bcl11b controlled expression of essential Treg program genes at steady state and in inflammation. Bcl11b bound to genomic regulatory regions of Treg program genes in both human and mouse Treg cells, overlapping with Foxp3 binding; these genes showed altered chromatin accessibility in the absence of Bcl11b. Additionally, Bcl11b restrained myeloid and NK cell programs in Treg cells. Our study provides new mechanistic insights on the Treg cell program and identity control, with major implications for therapies in autoimmunity and cancer.
Subject(s)
Autoimmunity , Fetus/immunology , Repressor Proteins/metabolism , T-Lymphocytes, Regulatory/metabolism , Tumor Suppressor Proteins/metabolism , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Lineage , Colitis/etiology , Colitis/immunology , Colitis/pathology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/mortality , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Forkhead Transcription Factors/metabolism , Humans , Kaplan-Meier Estimate , Male , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Binding , Repressor Proteins/genetics , Skin/pathology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , Tumor Suppressor Proteins/geneticsABSTRACT
During the last 3 decades, there has been a slow advance to obtain new treatments for malignant melanoma that improve patient survival. In this work, we present a systematic study focused on the antiproliferative and antitumour effect of AgNPs. These nanoparticles are fully characterized, are coated with polyvinylpyrrolidone (PVP), and have an average size of 35 ± 15 nm and a metallic silver content of 1.2% wt. Main changes on cell viability, induction of apoptosis and necrosis, and ROS generation were found on B16-F10 cells after six hours of exposure to AgNPs (IC50 = 4.2 µg/mL) or Cisplatin (IC50 = 2.0 µg/mL). Despite the similar response for both AgNPs and Cisplatin on antiproliferative potency (cellular viability of 53.95 ± 1.88 and 53.62 ± 1.04) and ROS production (20.27 ± 1.09% and 19.50 ± 0.35%), significantly different cell death pathways were triggered. While AgNPs induce only apoptosis (45.98 ± 1.88%), Cisplatin induces apoptosis and necrosis at the same rate (22.31 ± 1.72% and 24.07 ± 1.10%, respectively). In addition to their antiproliferative activity, in vivo experiments showed that treatments of 3, 6, and 12 mg/kg of AgNPs elicit a survival rate almost 4 times higher (P < 0.05) compared with the survival rate obtained with Cisplatin (2 mg/kg). Furthermore, the survivor mice treated with AgNPs do not show genotoxic damage determined by micronuclei frequency quantification on peripheral blood cells. These results exhibit the remarkable antitumour activity of a nongenotoxic AgNP formulation and constitute the first advance toward the application of these AgNPs for melanoma treatment, which could considerably reduce adverse effects provoked by currently applied chemotherapeutics.
Subject(s)
Melanoma, Experimental/drug therapy , Metal Nanoparticles/therapeutic use , Silver/chemistry , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/therapeutic use , DNA Damage/drug effects , Erythrocytes/cytology , Erythrocytes/drug effects , Erythrocytes/metabolism , Kaplan-Meier Estimate , Male , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Metal Nanoparticles/chemistry , Metal Nanoparticles/toxicity , Mice , Mice, Inbred C57BL , Reactive Oxygen Species/metabolismABSTRACT
Mammals evolved in the face of fluctuating food availability. How the immune system adapts to transient nutritional stress remains poorly understood. Here, we show that memory T cells collapsed in secondary lymphoid organs in the context of dietary restriction (DR) but dramatically accumulated within the bone marrow (BM), where they adopted a state associated with energy conservation. This response was coordinated by glucocorticoids and associated with a profound remodeling of the BM compartment, which included an increase in T cell homing factors, erythropoiesis, and adipogenesis. Adipocytes, as well as CXCR4-CXCL12 and S1P-S1P1R interactions, contributed to enhanced T cell accumulation in BM during DR. Memory T cell homing to BM during DR was associated with enhanced protection against infections and tumors. Together, this work uncovers a fundamental host strategy to sustain and optimize immunological memory during nutritional challenges that involved a temporal and spatial reorganization of the memory pool within "safe haven" compartments.
Subject(s)
Bone Marrow/metabolism , Immunologic Memory , Animals , Bone Marrow/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Caloric Restriction/veterinary , Cell Line, Tumor , Chemokine CXCL12/metabolism , Diet, Reducing/veterinary , Energy Metabolism , Gene Expression Regulation , Glucocorticoids , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-akt/metabolism , Receptors, CXCR4/metabolism , Survival Rate , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Myeloid cells contribute to tumor progression, but how the constellation of receptors they express regulates their functions within the tumor microenvironment (TME) is unclear. We demonstrate that Fcmr (Toso), the putative receptor for soluble IgM, modulates myeloid cell responses to cancer. In a syngeneic melanoma model, Fcmr ablation in myeloid cells suppressed tumor growth and extended mouse survival. Fcmr deficiency increased myeloid cell population density in this malignancy and enhanced anti-tumor immunity. Single-cell RNA sequencing of Fcmr-deficient tumor-associated mononuclear phagocytes revealed a unique subset with enhanced antigen processing/presenting properties. Conversely, Fcmr activity negatively regulated the activation and migratory capacity of myeloid cells in vivo, and T cell activation by bone marrow-derived dendritic cells in vitro. Therapeutic targeting of Fcmr during oncogenesis decreased tumor growth when used as a single agent or in combination with anti-PD-1. Thus, Fcmr regulates myeloid cell activation within the TME and may be a potential therapeutic target.