Phenotypic Reprogramming Research Articles

Crotoxin Modulates Macrophage Phenotypic Reprogramming.

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 μg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 μg/animal: M1 and at 5.0 μg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 μg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

Fatty Acid Metabolites and the Tumor Microenvironment as Potent Regulators of Cancer Stem Cell Signaling.

Individual cancer cells are not equal but are organized into a cellular hierarchy in which only a rare few leukemia cells can self-renew in a manner reminiscent of the characteristic stem cell properties. The PI3K/AKT pathway functions in a variety of cancers and plays a critical role in the survival and proliferation of healthy cells under physiologic conditions. In addition, cancer stem cells might exhibit a variety of metabolic reprogramming phenotypes that cannot be completely attributed to the intrinsic heterogeneity of cancer. Given the heterogeneity of cancer stem cells, new strategies with single-cell resolution will become a powerful tool to eradicate the aggressive cell population harboring cancer stem cell phenotypes. Here, this article will provide an overview of the most important signaling pathways of cancer stem cells regarding their relevance to the tumor microenvironment and fatty acid metabolism, suggesting valuable strategies among cancer immunotherapies to inhibit the recurrence of tumors.

Oncogenic metabolic reprogramming in breast cancer: focus on signaling pathways and mitochondrial genes.

Oncogenic metabolic reprogramming impacts the abundance of key metabolites that regulate signaling and epigenetics. Metabolic vulnerability in the cancer cell is evident from the Warburg effect. The research on metabolism in the progression and survival of breast cancer (BC) is under focus. Oncogenic signal activation and loss oftumor suppressor are important regulators of tumor cell metabolism. Several intrinsic and extrinsic factors contribute to metabolic reprogramming. The molecular mechanisms underpinning metabolic reprogramming in BC are extensive and only partially defined. Various signaling pathways involved in the metabolism play a significant role in the modulation of BC. Notably, PI3K/AKT/mTOR pathway, lactate-ERK/STAT3 signaling, loss of the tumor suppressor Ras, Myc, oxidative stress, activation of the cellular hypoxic response and acidosis contribute to different metabolic reprogramming phenotypes linked to enhanced glycolysis. The alterations in mitochondrial genes have also been elaborated upon along with their functional implications. The outcome of these active research areas might contribute to the development of novel therapeutic interventions and the remodeling of knowndrugs.

Smooth Muscle Cell Klf4 Expression Is Not Required for Phenotype Modulation or Aneurysm Formation in Marfan Syndrome Mice-Brief Report.

Smooth muscle cell (SMC) phenotypic reprogramming toward a mixed synthetic-proteolytic state is a central feature of aortic root aneurysm in Marfan syndrome (MFS). Previous work identified Klf4 as a potential mediator of SMC plasticity in MFS. MFS (Fbn1C1041G/+) mouse strains with an inducible vascular SMC fluorescent reporter (MFSSMC) with or without SMC-specific deletion of Klf4 exons 2 to 3 (MFSSMC-Klf4Δ) were generated. Simultaneous SMC tracing and Klf4 loss-of-function (Klf4Δ mice) was induced at 6 weeks of age. Aneurysm growth was assessed via serial echocardiography (4-24 weeks). Twenty-four-week-old mice were assessed via histology, RNA in situ hybridization, and aortic single-cell RNA sequencing. MFS mice demonstrated progressive aortic root dilatation compared with control (WTSMC) mice regardless of Klf4 genotype (P<0.001), but there was no difference in aneurysm growth in MFSSMC-Klf4Δ versus MFSSMC (P=0.884). Efficient SMC Klf4 deletion was confirmed via lineage-stratified genotyping, RNA in situ hybridization, and immunohistochemistry. Single-cell RNA sequencing of traced SMCs revealed a highly similar pattern of phenotype modulation marked by loss of contractile markers (eg, Myh11, Cnn1) and heightened expression of matrix genes (eg, Col1a1, Fn1) between Klf4 genotypes. Pseudotemporal quantitation of SMC dedifferentiation confirmed that Klf4 deletion did not alter the global extent of phenotype modulation, but reduced expression of 23 genes during this phenotype transition in MFSSMC-Klf4Δmice, including multiple chondrogenic genes expressed by only the most severely dedifferentiated SMCs (eg, Cytl1, Tnfrsf11b). Klf4 is not required to initiate SMC phenotype modulation in MFS aneurysm but may exert regulatory control over chondrogenic genes expressed in highly dedifferentiated SMCs.

Is Cancer Reversible? Rethinking Carcinogenesis Models-A New Epistemological Tool.

A growing number of studies shows that it is possible to induce a phenotypic transformation of cancer cells from malignant to benign. This process is currently known as "tumor reversion". However, the concept of reversibility hardly fits the current cancer models, according to which gene mutations are considered the primary cause of cancer. Indeed, if gene mutations are causative carcinogenic factors, and if gene mutations are irreversible, how long should cancer be considered as an irreversible process? In fact, there is some evidence that intrinsic plasticity of cancerous cells may be therapeutically exploited to promote a phenotypic reprogramming, both in vitro and in vivo. Not only are studies on tumor reversion highlighting a new, exciting research approach, but they are also pushing science to look for new epistemological tools capable of better modeling cancer.

High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias

Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution.

Anoikis in phenotypic reprogramming of the prostate tumor microenvironment.

Prostate cancer is one of the most common malignancies in males wherein 1 in 8 men are diagnosed with this disease in their lifetime. The urgency to find novel therapeutic interventions is associated with high treatment resistance and mortality rates associated with castration-resistant prostate cancer. Anoikis is an apoptotic phenomenon for normal epithelial or endothelial cells that have lost their attachment to the extracellular matrix (ECM). Tumor cells that lose their connection to the ECM can die via apoptosis or survive via anoikis resistance and thus escaping to distant organs for metastatic progression. This review discusses the recent advances made in our understanding of the signaling effectors of anoikis in prostate cancer and the approaches to translate these mechanistic insights into therapeutic benefits for reducing lethal disease outcomes (by overcoming anoikis resistance). The prostate tumor microenvironment is a highly dynamic landscape wherein the balance between androgen signaling, cell lineage changes, epithelial-mesenchymal transition (EMT), extracellular matrix interactions, actin cytoskeleton remodeling as well as metabolic changes, confer anoikis resistance and metastatic spread. Thus, these mechanisms also offer unique molecular treatment signatures, exploitation of which can prime prostate tumors to anoikis induction with a high translational significance.

MAIT cell inhibition promotes liver fibrosis regression via macrophage phenotype reprogramming

Recent data have shown that liver fibrosis can regress even at later stages of cirrhosis and shifting the immune response from pro-inflammatory towards a resolutive profile is considered as a promising option. The immune regulatory networks that govern the shift of the inflammatory phenotype and thus potential reversal of liver fibrosis are lesser known. Here we show that in precision-cut human liver slices obtained from patients with end-stage fibrosis and in mouse models, inhibiting Mucosal-Associated Invariant T (MAIT) cells using pharmacological or antibody-driven approaches, limits fibrosis progression and even regresses fibrosis, following chronic toxic- or non-alcoholic steatohepatitis (NASH)-induced liver injury. Mechanistic studies, combining RNA sequencing, in vivo functional studies (performed in male mice) and co-culture experiments indicate that disruption of the MAIT cell-monocyte/macrophage interaction results in resolution of fibrosis both by increasing the frequency of restorative Ly6Clo at the expenses of pro-fibrogenic Ly6Chi monocyte-derived macrophages and promoting an autophagic phenotype in both subsets. Thus, our data show that MAIT cell activation and the consequential phenotype shift of liver macrophages are important pathogenic features of liver fibrosis and could be targeted by anti-fibrogenic therapy.

Melanoma-associated repair-like Schwann cells suppress anti-tumor T-cells via 12/15-LOX/COX2-associated eicosanoid production

ABSTRACT Peripheral glia, specifically the Schwann cells (SCs), have been implicated in the formation of the tumor microenvironment (TME) and in cancer progression. However, in vivo and ex vivo analyses of how cancers reprogram SC functions in different organs of tumor-bearing mice are lacking. We generated Plp1-CreERT/tdTomato mice which harbor fluorescently labeled myelinated and non-myelin forming SCs. We show that this model enables the isolation of the SCs with high purity from the skin and multiple other organs. We used this model to study phenotypic and functional reprogramming of the SCs in the skin adjacent to melanoma tumors. Transcriptomic analyses of the peritumoral skin SCs versus skin SCs from tumor-free mice revealed that the former existed in a repair-like state typically activated during nerve and tissue injury. Peritumoral skin SCs also downregulated pro-inflammatory genes and pathways related to protective anti-tumor responses. In vivo and ex vivo functional assays confirmed immunosuppressive activities of the peritumoral skin SCs. Specifically, melanoma-reprogrammed SCs upregulated 12/15-lipoxygenase (12/15-LOX) and cyclooxygenase (COX)-2, and increased production of anti-inflammatory polyunsaturated fatty acid (PUFA) metabolites prostaglandin E2 (PGE2) and lipoxins A4/B4. Inhibition of 12/15-LOX or COX2 in SCs, or EP4 receptor on lymphocytes reversed SC-dependent suppression of anti-tumor T-cell activation. Therefore, SCs within the skin adjacent to melanoma tumors demonstrate functional switching to repair-like immunosuppressive cells with dysregulated lipid oxidation. Our study suggests the involvement of the melanoma-associated repair-like peritumoral SCs in the modulation of locoregional and systemic anti-tumor immune responses.

SLC3A2 promotes tumor-associated macrophage polarization through metabolic reprogramming in lung cancer.

Tumor-associated macrophages (TAMs) are one of the most abundant immunosuppressive cells in the tumor microenvironment and possess crucial functions in facilitating tumor progression. Emerging evidence indicates that altered metabolic properties in cancer cells support the tumorigenic functions of TAMs. However, the mechanisms and mediators the underly the cross-talk between cancer cells and TAMs remain largely unknown. In the present study, we revealed that high solute carrier family 3 member 2 (SLC3A2) expression in lung cancer patients was associated with TAMs and poor prognosis. Knockdown of SLC3A2 in lung adenocarcinoma cells impaired M2 polarization of macrophages in a coculture system. Using metabolome analysis, we identified that SLC3A2 knockdown altered the metabolism of lung cancer cells and changed multiple metabolites, including arachidonic acid, in the tumor microenvironment. More importantly, we showed that arachidonic acid was responsible for SLC3A2-mediated macrophage polarization in the tumor microenvironment to differentiate into M2 type both in vitro and in vivo. Our data illustrate previously undescribed mechanisms responsible for TAM polarization and suggest that SLC3A2 acts as a metabolic switch on lung adenocarcinoma cells to induce macrophage phenotypic reprogramming through arachidonic acid.

The role of AMPK in cancer metabolism and its impact on the immunomodulation of the tumor microenvironment.

Adenosine monophosphate-activated protein kinase (AMPK) is a key metabolic sensor that is pivotal for the maintenance of cellular energy homeostasis. AMPK contributes to diverse metabolic and physiological effects besides its fundamental role in glucose and lipid metabolism. Aberrancy in AMPK signaling is one of the determining factors which lead to the development of chronic diseases such as obesity, inflammation, diabetes, and cancer. The activation of AMPK and its downstream signaling cascades orchestrate dynamic changes in the tumor cellular bioenergetics. It is well documented that AMPK possesses a suppressor role in the context of tumor development and progression by modulating the inflammatory and metabolic pathways. In addition, AMPK plays a central role in potentiating the phenotypic and functional reprogramming of various classes of immune cells which reside in the tumor microenvironment (TME). Furthermore, AMPK-mediated inflammatory responses facilitate the recruitment of certain types of immune cells to the TME, which impedes the development, progression, and metastasis of cancer. Thus, AMPK appears to play an important role in the regulation of anti-tumor immune response by regulating the metabolic plasticity of various immune cells. AMPK effectuates the metabolic modulation of anti-tumor immunity via nutrient regulation in the TME and by virtue of its molecular crosstalk with major immune checkpoints. Several studies including that from our lab emphasize on the role of AMPK in regulating the anticancer effects of several phytochemicals, which are potential anticancer drug candidates. The scope of this review encompasses the significance of the AMPK signaling in cancer metabolism and its influence on the key drivers of immune responses within the TME, with a special emphasis on the potential use of phytochemicals to target AMPK and combat cancer by modulating the tumor metabolism.

Lactic acidosis induces metabolic and phenotypic reprogramming in cholangiocarcinoma cells via the upregulation of thrombospondin-1.

The high glycolytic activity of cancer cells leads to lactic acidosis (LA) in the tumor microenvironment. LA is not merely a consequence of metabolic activities but also has functional roles in metabolic reprogramming and cancer progression. Cholangiocarcinoma (CCA) cells exhibit a high dependency on glycolysis for survival and growth, but the specific effects of LA on cellular characteristics remain unknown. Here, we demonstrate that long-term LA (LLA) reprograms the metabolic phenotype of CCA cells from glycolytic to oxidative and enhances their migratory activity. In CCA cell culture, short-term LA (24 h) showed a growth inhibitory effect, while extended LA exposure for more than 2 weeks (LLA) led to enhanced cell motility. Coincidentally, LLA enhanced the respiratory capacity with an increase in mitochondrial mass. Inhibition of mitochondrial function abolished LLA-induced cell motility, suggesting that metabolic remodeling affects the phenotypic outcomes. RNA-sequencing analysis revealed that LLA upregulated genes associated with cell migration and epithelial-mesenchymal transition (EMT), including thrombospondin-1 (THBS1), which encodes a pro-EMT-secreted protein. Inhibition of THBS1 resulted in the suppression of both LLA-induced cell motility and respiratory capacity. Moreover, high THBS1 expression was associated with poor survival in patients with CCA. Collectively, our study suggests that the increased expression of THBS1 by LLA promotes phenotypic alterations, leading to CCA progression.

Метаболическая регуляция функций натуральных киллеров

Changes in the body metabolism affect the metabolic and functional activity of cells of the immune system. Pregnancy is characterized by changes in hormone production, basal metabolism, immunoreactivity, increase of fat mass. During pregnancy, NK cells acquire a regulatory phenotype, migrate to the uterus and become the dominant subpopulation of lymphocytes in uterus (decidual NK), which is necessary to maintain the invasive syncytiotrophoblast growth. This transformation into decidual NK cells is accompanied by increased glucose consumption and a predominant transition from oxidative phosphorylation to glycolysis. The key molecules involved in controlling the implementation of metabolic programs of NK cells are the target for rapamycin in mammalian cells (mTOR) and AMP-activated protein kinase (AMPK). The modularization of mTOR and AMPK activity by various agents (hormones, cytokines) during pregnancy determines the metabolic reprogramming of the NK cell phenotype and functions. The study of the metabolic regulation of the NK functional activity is necessary to increase the effectiveness of NK cell therapy.

Intracavitary adoptive transfer of IL-12 mRNA-engineered tumor-specific CD8+ T cells eradicates peritoneal metastases in mouse models

ABSTRACT Previous studies have shown that local delivery of tumor antigen-specific CD8+ T lymphocytes engineered to transiently express single-chain IL-12 mRNA is highly efficacious. Peritoneal dissemination of cancer is a frequent and often fatal patient condition usually diagnosed when the tumor burden is too large and hence uncontrollable with current treatment options. In this study, we have modeled intracavitary adoptive T cell therapy with OVA-specific OT-I T cells electroporated with IL-12 mRNA to treat B16-OVA and PANC02-OVA tumor spread in the peritoneal cavity. Tumor localization in the omentum and the effects of local T-cell encounter with the tumor antigens were monitored, the gene expression profile evaluated, and the phenotypic reprogramming of several immune subsets was characterized. Intraperitoneal administration of T cells promoted homing to the omentum more effectively than intravenous administration. Transient IL-12 expression was responsible for a favorable reprogramming of the tumor immune microenvironment, longer persistence of transferred T lymphocytes in vivo, and the development of immunity to endogenous antigens following primary tumor eradication. The efficacy of the strategy was at least in part recapitulated with the adoptive transfer of lower affinity transgenic TCR-bearing PMEL-1 T lymphocytes to treat the aggressive intraperitoneally disseminated B16-F10 tumor. Locoregional adoptive transfer of transiently IL-12-armored T cells appears to offer promising therapeutic advantages in terms of anti-tumor efficacy to treat peritoneal carcinomatosis.

Nanomaterials targeting macrophages in sepsis: A promising approach for sepsis management.

Sepsis is a life-threatening organ dysfunction resulting from dysregulated host responses to infection. Macrophages play significant roles in host against pathogens and the immunopathogenesis of sepsis, such as phagocytosis of pathogens, secretion of cytokines, and phenotype reprogramming. However, the rapid progression of sepsis impairs macrophage function, and conventional antimicrobial and supportive treatment are not sufficient to restore dysregulated macrophages roles. Nanoparticles own unique physicochemical properties, surface functions, localized surface plasmon resonance phenomenon, passive targeting in vivo, good biocompatibility and biodegradability, are accessible for biomedical applications. Once into the body, NPs are recognized by host immune system. Macrophages are phagocytes in innate immunity dedicated to the recognition of foreign substances, including nanoparticles, with which an immune response subsequently occurs. Various design strategies, such as surface functionalization, have been implemented to manipulate the recognition of nanoparticles by monocytes/macrophages, and engulfed by them to regulate their function in sepsis, compensating for the shortcomings of sepsis traditional methods. The review summarizes the mechanism of nanomaterials targeting macrophages and recent advances in nanomedicine targeting macrophages in sepsis, which provides good insight for exploring macrophage-based nano-management in sepsis.

Heme and Iron Aggravate Tissue Damage in Sickle Cell Disease By Driving Macrophage Functional Reprogramming through the Coordinated Suppression of Efferocytosis and Mitochondrial Metabolism

Introduction: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is in part driven by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme released upon hemolysis, which acts as damage-associated molecular pattern to stimulate macrophage inflammatory phenotypic switching through TLR4 signaling and ROS production (Vinchi et al., Blood 2016). Heme-induced pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to the inflammatory landscape and organ damage typically associated with this disease. While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties remains unexplored. Macrophage functions such as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages (BMDMs), to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme severely alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme excess drives defective efferocytosis of apoptotic neutrophils in BMDMs and mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the nuclear transcription factor PPARγ and its coactivator PGC1α, which lead to reduced expression of efferocytic receptors and impaired mitochondrial dynamics and functions. This results in limited recognition/engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4 and IL-10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. Heme-driven changes are associated with cell metabolic skewing towards glycolysis and pentose phosphate pathway and reduced reliance on mitochondrial fatty acid oxidation and TCA cycle. Macrophages pre-loaded with fatty acids showed decreased oxygen consumption rate and ATP production after heme exposure, indicating that heme-altered mitochondrial dynamics impair lipid catabolism via limited mitochondrial oxidative phosphorylation. Overall, heme induces macrophage phenotypic and functional reprogramming through the coordinated suppression of efferocytosis, via efferocytosis receptor downregulation, and fatty acid β-oxidation, via altered mitochondrial dynamics. We further demonstrate that heme-impaired efferocytosis and tissue damage promote autoimmunity through anti-nuclear antibody production against 'self' secondary necrotic cells, potentially explaining the increasing evidence of autoimmune diseases in SCD patients. Finally, hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment improve tissue damage in hemolytic mice through macrophage rewiring. Defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients' plasma and rescued by heme scavenging via haptoglobin/hemopexin and PPARγ/PGC1α modulation via PPARα agonist or IL-4. Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation significantly promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events and prevent autoimmune conditions.

Single-Cell Analyses of Immune Microenvironment Informs Potential Benefits of Dual Macrophage-Targeted Therapy in Multiple Myeloma

Background: Multiple myeloma(MM) remains incurable as most patients endure multiple progressions. The complexity of tumor-associated environment(TME) in the progression of MM was not fully clarified. We investigated the atlas of TME during the process of disease progression and explored potential novel therapeutic target. Methods: We performed in-depth single-cell RNA sequencing(scRNAseq) analysis on the bone marrow(BM) cells from 14 MM patients at different disease stage and validate the findings from scRNAseq analysis by bulk RNAseq, flow cytometry analysis and in vitro & in vivo functional experiments. Results: We delineated a compromised TME during progression, characterized by abnormal enrichment of NK cells and exhausted-CD8+T cells, and reprogramming of macrophages(MΦs). A significant enrichment in NK cells and a decrease in CD34+ hematopoietic cells were observed, and the enrichment in NK cells was associated with the progression of disease stage. We found CD8+T cells expressing LAG3 and TIGIT were enriched in advanced disease stage. We further dissected the gene expression signatures of myeloid lineage cells and identified five monocyte/macrophage subsets, which were further divided into CD14+subsets (hm1, hm4, hm5) and CD16+subsets (hm3, hm6) （FigureA). In previous studies reported by other researchers, macrophages presenting M2 phenotype were highly enriched in the BM microenvironment and contributed to the progression of MM. We calculated M1/M2 polarization score for monocyte/macrophage clusters (Figure B). The gene expression signatures of the five macrophage clusters did not fit with either the canonical M1 or M2 classifications, demonstrating a reprogrammed phenotype rather than the M1/M2 paradigm. However, the two CD14+subsets (hm4 and hm5) only present in myeloma stage demonstrated high M2 score than other macrophage clusters (hm1, hm3 and hm6), implying the potential role of M2 polarization in myeloma progression. TAMs reprogramming was correlated with MM progression, mainly involving the CD14+subsets identified in late-stage-disease, featured by a prone-M2 phenotype and phagocytic dysfunction. Transcription factors associated with macrophage M2 polarization and immunosuppression were preferentially expressed in different TAMs clusters. We validate the reprogrammed phenotype of macrophages found in scRNAseq data by flow cytometry analysis and bulk RNAseq in patients samples and in vitro cell experiments. To interrogate global cell-cell interactions in myeloma during disease progression, we performed computational modeling using 'CellPhoneDB' to identify interactions between different monocyte/macrophage subclusters and plasma cells (PCs) in the BM microenvironment from different stage disease(Figure C). We identified two significantly enriched ligand-receptor pairs including the SIRPA-CD47 'don't eat me' pathway suppressing phagocytosis and the CD74-MIF(macrophage inhibitory factor) reshaping the MΦs phenotype. As CD47 and MIF molecules are linked with disease progression and adverse outcomes, we proposed the hypothesis that dual immunosuppression of TAMs by CD47 and MIF promotes disease progression in MM. Finally, we designed a dual-MΦs-targeted therapeutic strategy by combining anti-CD47 mAbs and MIF inhibitor to activate phagocytosis and repolarize MΦ to a functional phenotype, and proved the potent anti-tumor effect in vitro and in vivo. Conclusion: We identified compositional alterations in MM-TME during the disease progression and unraveled significant TAMs phenotype reprogramming and phagocytic dysfunction. We discovered critical dual cellular cross-talks (CD47 and MIF) between MΦs and PCs, and explored their function and clinical significance. Finally, we designed a dual MΦs-targeted therapeutic strategy and proved its anti-tumor effect and the potential clinical benefits. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Experimental in vitro phenotype reprogramming of two subsets of neutrophilic granulocytes in children with acute destructive pneumonia by means of a synthetic hexapeptide

Effector dysfunctions of neutrophil granulocytes are often associated with the occurrence of dysregulatory processes in the antibacterial immune defense. Acute destructive pneumonia is a severe purulent-inflammatory disease associated with discordant functions of effector mechanisms of neutrophil granulocytes and emergence of negatively transformed cell subsets. Therefore, the search for new experimental approaches aimed at re-orientation of negatively altered phenotype of distinct subsets of neutrophilic granulocytes in the children with acute destructive pneumonia by means of various immunotropic substances is quite relevant. The aim of the study was to evaluate the modulating effects of synthetic hexapeptide (Arginyl-alpha-Aspartyl-Lysyl-Valyl-Tyrosyl-Arginine) on the contents and phenotype of 2 functionally significant subsets of major (CD16+CD64-CD32+CD11b+) and minor (CD16+CD64+CD32+CD11b+) subpopulations of neutrophils in a closed in vitro experimental system sampled in the children with atypical acute destructive pneumonia. We have examined twenty peripheral blood samples from 10 children with acute destructive pneumonia, and 40 blood samples of 20 healthy children 2-4 years old. Immunophenotyping of neutrophil granulocytes classified in 2 subsets was performed on the basis of expression density of membrane receptors, according to MFI criteria. Phenotypic features of neutrophil granulocyte subsets were evaluated in the in vitro system before and after incubation of peripheral blood with Hexapeptide (10-6 g/L; 37 C, 60 min). In children with acute destructive pneumonia, compared with conditionally healthy children, the following variants of negative transformation of the neutrophil subsets were established: a significant decrease in the ratios of the major subset, i.e., from 98.0 (96.9-98.7) % o 55.8 (35.3-74.8) %, with a decreased CD16 and CD11b density expression according to MFI, and a significantly increase ratio of the minor neutrophil subset: from 1.3 (0.4-1.6) % to 52.6 (41.8-54.9) %, with increased expression of CD11b receptor, and a decrease in CD64 expression. Immunomodulatory effects of Hexapeptide upon neutrophil granulocytes of children with acute destructive pneumonia have been demonstrated in the closed in vitro system showing positive phenotype remodeling of both cell subsets in the absence of significant quantitative changes. Thus, upon treatment with the hexapeptide, we have found a significantly increased expression of activation receptors CD16, CD11b in the major subset, and a significant decrease in their expression for the minor subset to the levels typical to healthy children. At the same time, hexapeptide did not affect the studied subsets of neutrophils from healthy children, except of increased CD64 expression in the minor subset. The obtained data can be used in future to develop new approaches to the targeted immunotherapy aimed at correcting the phenotype of neutrophil granulocyte subsets in acute destructive pneumonia in children.

Metabolic reprogramming and alteration of the redox state in hyper-productive MDCK cells for influenza a virus production

Influenza is a global public health issue leading to widespread morbidity and mortality with devastating economic loss annually. Madin-Darby Canine Kidney (MDCK) cell line has been a major cell line for influenza vaccine applications. Though many details of the host metabolic responses upon influenza A virus (IAV) infection have been documented, little is known about the metabolic reprogramming features of a hyper-productive host for IAV vaccine production. In this study, a MDCK cell clone H1 was shown to have a particular high productivity of 30 × 103 virions/cell. The glucose and amino acid metabolism of H1 were evaluated, indicating that the high producer had a particular metabolic reprogramming phenotype compared to its parental cell line (P): elevated glucose uptake, superior tricarboxylic acid cycle flux, moderate amino acid consumption, and better regulation of reactive oxygen species. Combined with the stronger mitochondrial function and mild antiviral and inflammatory responses characterized previously, our results indicated that the high producer had a sufficient intracellular energy supply, and balanced substrate distribution for IAV and host protein synthesis as well as the intracellular redox status. Understanding of these metabolic alterations paves the way for the rational cell line development and reasonable process optimization for high-yield influenza vaccine production.

Extracellular Vesicles-ceRNAs as Ovarian Cancer Biomarkers: Looking into circRNA-miRNA-mRNA Code.

Simple SummaryPatients with ovarian cancer have a very poor chance of long-term survival, usually due to advanced disease at the time of diagnosis. Emerging evidence suggests that extracellular vesicles contain noncoding RNAs such as microRNAs, piwiRNAs, circular RNAs, and long noncoding RNAs, with regulatory effects on ovarian cancer. In this review, we focus on ovarian cancer-associated circular RNA shuttled by extracellular vesicles as mediators of cancer progression and novel biomarkers in liquid biopsy. We propose a circular-RNA–microRNA-mRNA code that can reveal the regulatory network created by extracellular vesicles, noncoding RNAs, and mRNAs in ovarian cancer. Future research in this field will help to identify novel diagnostic biomarkers and druggable therapeutic targets, which will ultimately benefit patients.Ovarian cancer (OC) is one of the most lethal gynecologic malignancies in females worldwide. OC is frequently diagnosed at an advanced stage due to a lack of specific symptoms and effective screening tests, resulting in a poor prognosis for patients. Age, genetic alterations, and family history are the major risk factors for OC pathogenesis. Understanding the molecular mechanisms underlying OC progression, identifying new biomarkers for early detection, and discovering potential targets for new drugs are urgent needs. Liquid biopsy (LB), used for cancer detection and management, consists of a minimally invasive approach and practical alternative source to investigate tumor alterations by testing extracellular vesicles (EVs), circulating tumor cells, tumor-educated platelets, and cell-free nucleic acids. EVs are nanosize vesicles shuttling proteins, lipids, and nucleic acids, such as DNA, RNA, and non-coding RNAs (ncRNAs), that can induce phenotypic reprogramming of target cells. EVs are natural intercellular shuttles for ncRNAs, such as microRNAs (miRNAs) and circular-RNAs (circRNAs), known to have regulatory effects in OC. Here we focus on the involvement of circRNAs and miRNAs in OC cancer progression. The circRNA-microRNA-mRNA axis has been investigated with Circbank and miRwalk analysis, unraveling the intricate and detailed regulatory network created by EVs, ncRNAs, and mRNAs in OC.