ABSTRACT
The proceeding study aimed to isolate glyphosate-degrading bacteria from soil and determine optimal degradation conditions through single-factor experiments and response surface methodology. The detoxifying efficacy of the isolate on glyphosate was assessed using earthworm model. The results indicate that Pseudomonas putida HE exhibited the highest glyphosate degradation rate. Optimal conditions for glyphosate degradation were observed at an inoculation percentage of approximately 5%, a pH of 7, and a temperature of 30 °C. Glyphosate induced notable neurotoxicity and reproductive toxicity in earthworms, evidenced by reduced activity of the neurotoxicity-associated enzyme AChE. Additionally, an increase in the activities of catalase, superoxide dismutase, and lactate dehydrogenase was observed. H&E staining revealed structural disruptions in the earthworm clitellum, with notable atrophy in the structure of spermathecae. Furthermore, glyphosate activation of earthworm immune systems led to increased expression of immune-related genes, specifically coelomic cytolytic factor and lysozyme. Notably, the introduction of strain HE mitigated the glyphosate toxicity to the earthworms mentioned above. P. putida HE was able to increase soil enzyme activities that were reduced due to glyphosate. The isolate P. putida HE, emerged as an effective and cost-efficient remedy for glyphosate degradation and toxicity reduction in natural settings, showcasing potential applications in real ecological settings.
ABSTRACT
Oral opportunistic pathogen Fusobacterium nucleatum can participate in various disease processes through the metabolite hydrogen sulfide, such as halitosis and colorectal cancer. The object of this study is to identify inhibitor capable of suppressing Fn1220, which is the principal hydrogen sulfide-producing enzyme in F. nucleatum. Through this inhibition, we aim to reduce the hydrogen sulfide production of F. nucleatum, consequently diminishing its virulence. Employing molecular docking techniques for inhibitor screening, we identified dencichine as the monomeric compound from Chinese medicine exhibiting the lowest binding energy to Fn1220 among a set of 27,045 candidates, and evaluated in vitro the ability of dencichine to inhibit hydrogen sulfide production using bismuth chloride method. Additionally, we investigated its impact on key virulence factors, including biofilm formation, hemolysis, and adhesion factors of F. nucleatum, using the crystalline violet method, sheep blood method, and RT-qPCR, respectively. Furthermore, we assessed the influence of dencichine on the lifespan of Caenorhabditis elegans. Results showed that dencichine was a suitable inhibitor of the Fn1220 of F. nucleatum, which significantly inhibited the production of virulence factors, e.g., biofilm, hemolysin, FadA, and Fap2 of F. nucleatum and improved the survival of C. elegans. We successfully identified the inhibitor of the enzyme Fn1220, dencichine, which inhibited the production of hydrogen sulfide and attenuated the virulence of F. nucleatum and holds promise as a potential therapeutic avenue for addressing oral diseases, e.g., halitosis in the future.
ABSTRACT
Colorectal cancer is the third most common cancer in the world today, and studies have shown that the ratio of Candida to Saccharomyces cerevisiae increased, and the abundance of S. cerevisiae in the intestines of patients with colorectal cancer decreased, which suggests that there is an imbalance in the proportion of fungi in the intestines of patients with colorectal cancer. The objective of this study was to screen S. cerevisiae isolate from traditional Chinese fermentation starters and assess its ability to ameliorate dysbiosis and to alleviate the carcinogenic process of azoxymethane/dextran sodium sulfate-induced colorectal cancer in mice model. S. cerevisiae strain SC-2201 was isolated and exhibited probiotic properties, including the ability to survive in an acidic pH environment and in the presence of bile salts in the gastrointestinal tract, as well as antioxidant activities. Oral administration of S. cerevisiae SC-2201 not only alleviated weight loss but also reduced colonic shortening and histological damage in azoxymethane/dextran sodium sulfate-induced colorectal cancer in mice. Furthermore, the administration of S. cerevisiae SC-2201 suppressed the expression of proinflammatory mediators, such as interleukin-1ß, interleukin-6, cyclooxygenase-2, vascular endothelial growth factor, nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3. Specifically, the analysis of gut bacteriome showed a significant decrease in Bacteroidota and Campylobacterota levels, as well as an increase in Proteobacteria level in the colorectal cancer group, which was alleviated by supplementation with S. cerevisiae SC-2201. The analysis of the mycobiome revealed a significant increase in the levels of Basidiomycota, Apiosordaria, Naganishia, and Taphrina genera in the colorectal cancer group, which were alleviated after supplementation with S. cerevisiae SC-2201. However, the levels of Xenoramularia, Entoloma, and Keissleriella were significantly increased after administration with S. cerevisiae SC-2201. Overall, the findings of this study demonstrate that S. cerevisiae SC-2201 possesses potential probiotic properties and can effectively attenuate the development of colorectal cancer, highlighting its cancer-preventive potential. This is the first report of a S. cerevisiae strain isolated from traditional Chinese fermentation starters which showed good probiotic properties, and mitigated azoxymethane/dextran sodium sulfate-induced colorectal cancer by modulating the gut microbiome and blocking proinflammatory mediators in mice.
ABSTRACT
High concentrations of reactive oxygen and nitrogen species (RONS) are key characteristics of inflammatory sites. Scavenging RONS at the site of inflammation is an effective therapeutic strategy. This study introduces ultrasmall Cu2O@His nanoparticles with RONS-scavenging ability for the treatment of inflammatory bowel disease (IBD) in mice. The strong coordination between the nitrogen atom in histidine (His) and copper enhances the dispersion and stability of Cu2O@His. Due to their small size and large surface area, Cu2O@His exhibits outstanding RONS-clearing ability. Importantly, Cu2O@His can target mitochondrial sites and repair damaged mitochondria. With excellent dispersion and scavenging RONS ability, Cu2O@His demonstrates good efficacy in treating mouse IBD. This work provides a new paradigm for developing nanozymes with an ultrasmall size and multiple scavenging RONS abilities.
Subject(s)
Copper , Inflammatory Bowel Diseases , Mice , Animals , Histidine , Anti-Inflammatory Agents , Reactive Oxygen Species , Reactive Nitrogen Species , NitrogenABSTRACT
IMPORTANCE: Colorectal cancer (CRC) is the second most common cancer in the world; the main treatment for CRC is immunosuppressive therapy, but this therapy is only effective for a small percentage of CRC patients, so there is an urgent need for a treatment with fewer side effects and higher efficacy. This study demonstrated that Fusobacterium nucleatum with increased abundance in CRC can regulate the autophagy process and disrupt normal intestinal microbiota by producing hydrogen sulfide, factors that may be involved in the development and progression of CRC. This study may provide a reference for future CRC treatment options that are efficient and have fewer side effects.
Subject(s)
Colorectal Neoplasms , Gastrointestinal Microbiome , Hydrogen Sulfide , Humans , Fusobacterium nucleatum/physiology , Hydrogen Sulfide/pharmacology , AutophagyABSTRACT
BACKGROUND: Prostate cancer (PCa) remains the most common malignant tumor in men, and the clinical treatment still faces many challenges. Several molecular biomarkers of PCa progression have been reported, however, whether FOXS1 can serve as a new biomarker in PCa remains unknown. METHODS: FOXS1 and Gli1 expression was assessed by RT-qPCR and western blot. The binding and regulation roles between FOXS1 and Gli1 were confirmed by Co-IP and ubiquitination assays. Cell viability, proliferation, apoptosis, migration, invasion and EMT progress were assessed through CCK-8, colony formation, flow cytometry, wound-healing, transwell and western blot assays, respectively. In vivo nude mice tumorigenesis model was also conducted to verify PCa growth. RESULTS: FOXS1 was upregulated in the PCa TCGA dataset and cells. High FOXS1 level was correlated with PCa patients' worse tumor stage and shorter survival. FOXS1 knockdown inhibited PCa cell proliferation, invasion, migration, EMT and tumor growth while increased cell apoptosis. Furthermore, FOXS1 knockdown decreased the inactivation of Hedgehog (Hh) pathway. FOXS1 bind to Gli1 and decreased the ubiquitination of Gli1, which resulted in the upregulation of Gli1. Besides, both Gil1 overexpression and Hh signal activation reversed the suppression function of FOXS1 silencing on PCa growth and metastasis. CONCLUSION: FOXS1 bind and stabilized Gli1 by blocking Gli1 ubiquitination, thereby activating Hh signaling to promote PCa cell growth and metastasis.
Subject(s)
Hedgehog Proteins , Prostatic Neoplasms , Animals , Humans , Male , Mice , Cell Line, Tumor , Cell Movement , Cell Proliferation , Forkhead Transcription Factors/metabolism , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Mice, Nude , Prostatic Neoplasms/genetics , Zinc Finger Protein GLI1/genetics , Zinc Finger Protein GLI1/metabolismABSTRACT
Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8+ memory T cell progenitors that can become either functional stem-like T (TSTEM) cells or dysfunctional T progenitor exhausted (TPEX) cells. To that end, we demonstrated that TSTEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate TSTEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, TSTEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4+ T cells during TSTEM-like CAR-T cell production. Adoptive transfer of TSTEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of TSTEM-like CAR-T cells and an increased memory T cell pool. Last, TSTEM-like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8+CAR+ T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated TSTEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.
Subject(s)
Immunotherapy, Adoptive , Neoplasms , Humans , Immunotherapy, Adoptive/methods , T-Lymphocytes , Cytokines/metabolism , Stem Cells/metabolism , Receptors, Antigen, T-Cell/metabolismABSTRACT
The biocatalytic asymmetric reduction of prochiral ketones for the production of enantiopure alcohols is highly desirable due to its inherent advantages over chemical methods. In this study, a new bacterial strain capable of transforming ketones to corresponding alcohols with high activity and excellent enantioselectivity was discovered in a soil sample. The strain was subsequently identified as Bacillus cereus TQ-2 based on its physiological characteristics and 16S rDNA sequence analysis. Under optimized reaction conditions, the resting cells of B. cereus TQ-2 converted acetophenone to enantioenriched (R)-1-phenylethanol with 99% enantiometric excess following anti-Prelog's rule, which is scarce in biocatalytic ketone reduction. The optimum temperature for the cells was 30 °C, and considerable catalytic activity was observed over a broad pH range from 5.0 to 9.0. The cells showed enhanced catalytic activity in the presence of 15% (v/v) glycerol as a co-substrate. The catalytic activity can also be substantially improved by adding Ca2+ or K+ ions. Moreover, the B. cereus TQ-2 cell was highly active in reducing several structurally diverse ketones and aldehydes to form corresponding alcohols with good to excellent conversion. Our study provides a versatile whole-cell biocatalyst that can be used in the asymmetric reduction of ketones for the production of chiral alcohol, thereby expanding the biocatalytic toolbox for potential practical applications.
Subject(s)
Alcohols , Ketones , Oxidation-Reduction , Biocatalysis , Ethanol , Glycerol , StereoisomerismABSTRACT
In the era of sustainable development, the use of cell factories to produce various compounds by fermentation has attracted extensive attention; however, industrial fermentation requires not only efficient production strains, but also suitable extracellular conditions and medium components, as well as scaling-up. In this regard, the use of biological models has received much attention, and this review will provide guidance for the rapid selection of biological models. This paper first introduces two mechanistic modeling methods, kinetic modeling and constraint-based modeling (CBM), and generalizes their applications in practice. Next, we review data-driven modeling based on machine learning (ML), and highlight the application scope of different learning algorithms. The combined use of ML and CBM for constructing hybrid models is further discussed. At the end, we also discuss the recent strategies for predicting bioreactor scale-up and culture behavior through a combination of biological models and computational fluid dynamics (CFD) models.
ABSTRACT
BACKGROUND: Vaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8+Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate with longer survival in metastatic cutaneous melanoma. However, their capacity to respond to VM and their neoantigens is not known. METHODS: Using longitudinal samples, we explored the evolution of VM mutations by whole-exome sequencing and RNAseq, we also defined the immune context using multiplex immunohistochemistry and nanostring pan cancer immune profile. Then using fresh single cell suspensions of the metastatic samples, we explored VM T cells via mass cytometry and single cell RNAseq and T cell receptor sequencing (TCRseq). Finally, we investigated TRM, pre-TRM and exhausted T cell function against melanoma neo-antigens and melanoma differentiation antigens in vitro. RESULTS: Primary VM was non-inflamed and devoid of CD8+ TRM cells. In contrast, both metastases showed proliferating CD8+ TRM were clustered at the tumor margin, with increased numbers in the second ICB-refractory metastasis. The first metastasis showed dense infiltration of CD8+ T cells, the second showed immune exclusion with loss of melanoma cell Major histocompatibility complex (MHC)-I expression associated with downregulation of antigen presentation pathway gene expression. CD8+ TRM from both metastases responded to autologous melanoma cells more robustly than all other CD8+ T cell subsets. In addition, CD8+ TRM shared TCR clones across metastases, suggesting a response to common antigens, which was supported by recognition of the same neoantigen by expanded tumor infiltrating lymphocytes. CONCLUSIONS: In this study, we identified TRM clusters in VM metastases from a patient, but not primary disease. We showed TRM location at the tumor margin, and their superior functional response to autologous tumor cells, predicted neoantigens and melanoma differentiation antigens. These CD8+ TRM exhibited the highest tumor-responsive potential and shared their TCR with tumor-infiltrating effector memory T cells. This suggests VM metastases from this patient retain strong antitumor T cell functional responses; however, this response is suppressed in vivo. The loss of VG MHC-I expression is a common immune escape mechanism which was not addressed by anti-PD-1 monotherapy; rather an additional targeted approach to upregulate MHC-I expression is required.
Subject(s)
Melanoma , Skin Neoplasms , CD8-Positive T-Lymphocytes , Female , Humans , Immune Checkpoint Inhibitors , Immunologic Memory , Memory T Cells , Skin Neoplasms/metabolismABSTRACT
Objective: Neuraminidase 1 (NEU1) participates in the response to multiple receptor signals and regulates various cellular metabolic behaviors. Importantly, it is closely related to the occurrence and progression of cardiovascular diseases. Because ischemic heart disease is often accompanied by impaired mitochondrial energy metabolism and oxidative stress. The purpose of this study was to investigate the functions and possible mechanisms of NEU1 in myocardial remodeling and mitochondrial metabolism induced by myocardial infarction (MI). Methods: In this study, the MI-induced mouse mode, hypoxia-treated H9C2 cells model, and hypoxia-treated neonatal rat cardiomyocytes (NRCMs) model were constructed. Echocardiography and histological analysis were adopted to evaluate the morphology and function of the heart at the whole heart level. Western blot was adopted to determine the related expression level of signaling pathway proteins and mitochondria. Mitochondrial energy metabolism and oxidative stress were detected by various testing kits. Results: Neuraminidase 1 was markedly upregulated in MI cardiac tissue. Cardiomyocyte-specific NEU1 deficiency restored cardiac function, cardiac hypertrophy, and myocardial interstitial fibrosis. What is more, cardiomyocyte-specific NEU1 deficiency inhibited mitochondrial dysfunction and oxidative stress induced by MI. Further experiments found that the sirtuin-1/peroxisome proliferator-activated receptor γ coactivator α (SIRT1/PGC-1α) protein level in MI myocardium was down-regulated, which was closely related to the above-mentioned mitochondrial changes. Cardiomyocyte-specific NEU1 deficiency increased the expression of SIRT1, PGC-1α, and mitochondrial transcription factor A (TFAM); which improved mitochondrial metabolism and oxidative stress. Inhibition of SIRT1 activity or PGC-1α activity eliminated the beneficial effects of cardiomyocyte-specific NEU1 deficiency. PGC-1α knockout mice experiments verified that NEU1 inhibition restored cardiac function induced by MI through SIRT1/PGC-1α signaling pathway. Conclusion: Cardiomyocyte-specific NEU1 deficiency can alleviate MI-induced myocardial remodeling, oxidative stress, and mitochondrial energy metabolism disorder. In terms of mechanism, the specific deletion of NEU1 may play a role by enhancing the SIRT1/PGC-1α signaling pathway. Therefore, cardiomyocyte-specific NEU1 may provide an alternative treatment strategy for heart failure post-MI.
ABSTRACT
BACKGROUND: Patients with BRAF-mutant and wild-type melanoma have different response rates to immune checkpoint blockade therapy. However, the reasons for this remain unknown. To address this issue, we investigated the precise immune composition resulting from BRAF mutation in treatment-naive melanoma to determine whether this may be a driver for different response to immunotherapy. METHODS: In this study, we characterized the treatment-naive immune context in patients with BRAF-mutant and BRAF wild-type (BRAF-wt) melanoma using data from single-cell RNA sequencing, bulk RNA sequencing, flow cytometry and immunohistochemistry (IHC). RESULTS: In single-cell data, BRAF-mutant melanoma displayed a significantly reduced infiltration of CD8+ T cells and macrophages but also increased B cells, natural killer (NK) cells and NKT cells. We then validated this finding using bulk RNA-seq data from the skin cutaneous melanoma cohort in The Cancer Genome Atlas and deconvoluted the data using seven different algorithms. Interestingly, BRAF-mutant tumors had more CD4+ T cells than BRAF-wt samples in both primary and metastatic cohorts. In the metastatic cohort, BRAF-mutant melanoma demonstrated more B cells but less CD8+ T cell infiltration when compared with BRAF-wt samples. In addition, we further investigated the immune cell infiltrate using flow cytometry and multiplex IHC techniques. We confirmed that BRAF-mutant melanoma metastases were enriched for CD4+ T cells and B cells and had a co-existing decrease in CD8+ T cells. Furthermore, we then identified B cells were associated with a trend for improved survival (p=0.078) in the BRAF-mutant samples and Th2 cells were associated with prolonged survival in the BRAF-wt samples. CONCLUSIONS: In conclusion, treatment-naive BRAF-mutant melanoma has a distinct immune context compared with BRAF-wt melanoma, with significantly decreased CD8+ T cells and increased B cells and CD4+ T cells in the tumor microenvironment. These findings indicate that further mechanistic studies are warranted to reveal how this difference in immune context leads to improved outcome to combination immune checkpoint blockade in BRAF-mutant melanoma.
Subject(s)
Melanoma , Proto-Oncogene Proteins B-raf , Skin Neoplasms , B-Lymphocytes , CD8-Positive T-Lymphocytes , Humans , Killer Cells, Natural , Melanoma/drug therapy , Melanoma/genetics , Melanoma/immunology , Mutation , Natural Killer T-Cells , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Tumor Microenvironment/geneticsABSTRACT
Lung cancer remains the most common fatal malignant disease, and the 5-year survival rate of patients with metastasis is merely 6%. In this research, the platinum nanocluster (short for nano-Pt) was used for optical imaging without the help of other fluorescent probes and possess targeted antitumor activity as well as low systemic toxicity. The endocytic pathway and distribution of nano-Pt in non-small cell lung cancer NSCLC H1299 cells was explored by the means of quantitative and qualitative tests. Furthermore, the targeting capability and antitumor efficiency of nano-Pt was detected by intravital imaging experiment and antitumor experiment. The research implies that nano-Pt entered H1299 cells dominatingly through macropinocytosis and clathrin-dependent endocytosis pathway, and has significant antitumor efficiency, targeting properties and reliable safety for mouse tumor, indicating this nano-Pt has great potential for clinical diagnosis and therapy of NSCLC H1299 cells.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Endocytosis , Humans , Lung Neoplasms/pathology , Mice , PlatinumABSTRACT
This study aims to explore the effects of chemical ingredient groups B and C in Kansui Radix stir-fried with vinegar on the diversity of gut microbiota in the rat model of malignant ascites, identify the key differential microbial taxa, and reveal the biological mechanism of water-expelling effect of the two chemical ingredient groups. The rat model of malignant ascites induced by Walker-256 cells was established, and phenolphthalein was used as the positive drug. The rats were orally administrated with corresponding agents for consecutive 7 days. On day 6, fresh feces samples were collected from the rats, and 16 S rDNA high-throughput sequencing and GC-MS were employed to determine the composition of gut microbiota and the content of short-chain fatty acids, respectively. On day 7, serum and intestinal tissue samples were collected for the determination of related indicators. Compared with the control group, the model group showed decreased feces volume and urine volume(P<0.01), increased volume of ascites and levels of Na~+, K~+, and Cl~- in urine(P<0.01), down-regulated mRNA and protein levels of intestinal AQP8(P<0.01), lowered abundance of beneficial Lactobacillus(P<0.01) while risen abundance of potential pathogenic Lachnospiraceae and Anaeroplasma(P<0.01), and reduced content of short-chain fatty acids(P<0.01). Compared with the model group, administration with chemical ingredient groups B and C alleviated all the above indicators(P<0.01). In conclusion, chemical ingredient groups B and C in Kansui Radix stir-fried with vinegar could alleviate the disordered gut microbiota in rats with malignant ascites to expel water through increasing the abundance of beneficial Lactobacillus and reducing the abundance of harmful Lachnospiraceae and Anaeroplasma. This study can provide a reference for the reasonable clinical application of Kansui Radix stir-fried with vinegar.
Subject(s)
Euphorbia , Gastrointestinal Microbiome , Acetic Acid/chemistry , Animals , Ascites/drug therapy , Euphorbia/chemistry , Plant Roots/chemistry , RatsABSTRACT
Diabetic cardiomyopathy (DCM) is associated with oxidative stress and augmented inflammation in the heart. Neuraminidases (NEU) 1 has initially been described as a lysosomal protein which plays a role in the catabolism of glycosylated proteins. We investigated the role of NEU1 in the myocardium in diabetic heart. Streptozotocin (STZ) was injected intraperitoneally to induce diabetes in mice. Neonatal rat ventricular myocytes (NRVMs) were used to verify the effect of shNEU1 in vitro. NEU1 is up-regulated in cardiomyocytes under diabetic conditions. NEU1 inhibition alleviated oxidative stress, inflammation and apoptosis, and improved cardiac function in STZ-induced diabetic mice. Furthermore, NEU1 inhibition also attenuated the high glucose-induced increased reactive oxygen species generation, inflammation and, cell death in vitro. ShNEU1 activated Sirtuin 3 (SIRT3) signaling pathway, and SIRT3 deficiency blocked shNEU1-mediated cardioprotective effects in vitro. More importantly, we found AMPKα was responsible for the elevation of SIRT3 expression via AMPKα-deficiency studies in vitro and in vivo. Knockdown of LKB1 reversed the effect elicited by shNEU1 in vitro. In conclusion, NEU1 inhibition activates AMPKα via LKB1, and subsequently activates sirt3, thereby regulating fibrosis, inflammation, apoptosis and oxidative stress in diabetic myocardial tissue.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Diabetes Mellitus, Experimental/complications , Diabetic Cardiomyopathies/genetics , Mucolipidoses/complications , Neuraminidase/genetics , Animals , Animals, Newborn , Apoptosis , Diabetes Mellitus, Experimental/genetics , Fibrosis , Inflammation , Male , Mice , Mice, Inbred C57BL , Mucolipidoses/genetics , Myocardium/pathology , Oxidative Stress , Rats , Signal Transduction , Sirtuin 3/metabolism , StreptozocinABSTRACT
PURPOSE: In this article, we describe a combination chimeric antigen receptor (CAR) T-cell therapy that eradicated the majority of tumors in two immunocompetent murine pancreatic cancer models and a human pancreatic cancer xenograft model. EXPERIMENTAL DESIGN: We used a dual-specific murine CAR T cell that expresses a CAR against the Her2 tumor antigen, and a T-cell receptor (TCR) specific for gp100. As gp100 is also known as pMEL, the dual-specific CAR T cells are thus denoted as CARaMEL cells. A vaccine containing live vaccinia virus coding a gp100 minigene (VV-gp100) was administered to the recipient mice to stimulate CARaMEL cells. The treatment also included the histone deacetylase inhibitor panobinostat (Pano). RESULTS: The combination treatment enabled significant suppression of Her2+ pancreatic cancers leading to the eradication of the majority of the tumors. Besides inducing cancer cell apoptosis, Pano enhanced CAR T-cell gene accessibility and promoted CAR T-cell differentiation into central memory cells. To test the translational potential of this approach, we established a method to transduce human T cells with an anti-Her2 CAR and a gp100-TCR. The exposure of the human T cells to Pano promoted a T-cell central memory phenotype and the combination treatment of human CARaMEL cells and Pano eradicated human pancreatic cancer xenografts in mice. CONCLUSIONS: We propose that patients with pancreatic cancer could be treated using a scheme that contains dual-specific CAR T cells, a vaccine that activates the dual-specific CAR T cells through their TCR, and the administration of Pano.
Subject(s)
Pancreatic Neoplasms , Receptors, Chimeric Antigen , Animals , Cell Line, Tumor , Histone Deacetylase Inhibitors/pharmacology , Humans , Immunotherapy, Adoptive/methods , Mice , Pancreatic Neoplasms/therapy , Panobinostat , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes , Xenograft Model Antitumor AssaysABSTRACT
Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.
Subject(s)
Antibodies/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Immune Checkpoint Inhibitors/pharmacology , Interleukin-33/pharmacology , Lymphocytes/drug effects , Melanoma, Experimental/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Skin Neoplasms/drug therapy , Animals , Cell Line, Tumor , Chemotaxis, Leukocyte/drug effects , Cytotoxicity, Immunologic/drug effects , Eosinophils/drug effects , Eosinophils/immunology , Eosinophils/metabolism , Female , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Lymphocytes/immunology , Lymphocytes/metabolism , Male , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/metabolism , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Skin Neoplasms/metabolismABSTRACT
Here we describe the application of mass cytometry to analyze tumor-infiltrating lymphocytes in human melanoma. Mass cytometry is the coupling of flow cytometry and mass spectrometry, which allows for the simultaneous measurement of 40+ cell parameters on a per cell basis. Heavy metal-labeled antibodies can bind to proteins (CD markers, transcription factors, cytokines) on the cell surface and in the cytoplasm/nucleus. As labeled cells pass through the CyTOF, the instrument detects the heavy metals. Combining these signals allows description of melanoma tumor-infiltrating lymphocytes at a greater depth than alternative phenotyping strategies and enables detailed analyses of a variety of cellular parameters, including immune cell lineage, activation status, and functional polarization.
Subject(s)
Flow Cytometry , Lymphocytes, Tumor-Infiltrating , Mass Spectrometry , Melanoma , Single-Cell Analysis , Humans , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Melanoma/metabolism , Melanoma/pathologyABSTRACT
Tumor-infiltrating lymphocytes (TILs) are an important prognostic indicator in melanoma and play a key role in the patient's response to immune checkpoint blockade. However, until recently, it was not possible to combine multi-parameter markers to define the TILs and their histological context. Multiplex immunohistochemistry (mIHC) is a new technology which addresses this issue and enables simultaneous detection of melanoma and multiple immune subsets in formalin fixed paraffin embedded tissue. Following antigen retrieval, melanoma tissue sections are stained by OPAL on an autostainer, including serial rounds of epitope labelling with monoclonal antibodies followed by tyramide signal amplification (TSA). The stained tissue sections are then imaged on the Vectra instrument, and digital images are processed by analysis software (inForm and HALO) to derive tissue segmentation and immune subset densities within the tumor and tumor stroma. Spatial relationships between immune cells and tumor cells are then analyzed using a novel R algorithm. Taken together, multiplex IHC describes the histological context of the immune system in melanoma. The data is objective and allows for characterization of individual melanomas as T cell inflamed (hot), immune excluded, or no immune cells (cold).