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Medical Oncology (Northwood, London,... Aug 2023The prostate cancer tumor microenvironment (TME) is comprised of many cell types that can contribute to and influence tumor progression. Some of the most abundant...
The prostate cancer tumor microenvironment (TME) is comprised of many cell types that can contribute to and influence tumor progression. Some of the most abundant prostate cancer TME cells are macrophages, which can be modeled on a continuous spectrum of M1-like (anti-tumor macrophages) to M2-like (pro-tumor macrophages). A function of M2-like macrophages is efferocytosis, the phagocytosis of apoptotic cells. Based on literature from other models and contexts, efferocytosis further supports the M2-like macrophage phenotype. MerTK is a receptor tyrosine kinase that mediates efferocytosis by binding phosphatidylserine on apoptotic cells. We hypothesize efferocytosis in the prostate cancer TME is a tumor-promoting function of macrophages and that targeting MerTK-mediated efferocytosis will slow prostate cancer growth and promote an anti-tumor immune infiltrate. The aims of this study are to measure efferocytosis of prostate cancer cells by in vitro human M1/M2 macrophage models and assess changes in the M2-like, pro-tumor macrophage phenotype following prostate cancer efferocytosis. Additionally, this study aims to demonstrate that targeting MerTK decreases prostate cancer efferocytosis and promotes an anti-tumor immune infiltrate. We have developed methodology using flow cytometry to quantify efferocytosis of human prostate cancer cells using the LNCaP cell line. We observed that M2 macrophages efferocytose the LNCaP cell line more than M1 macrophages. Following efferocytosis of LNCaP cells by M2 human monocyte-derived macrophages (HMDMs), we observed an increase in the M2-like, pro-tumor phenotype by flow cytometry cell surface marker analysis. By qRT-PCR, flow cytometry, and Western blot, we detected greater MerTK expression in M2 than M1 macrophages. Targeting MerTK with antibody Mer590 decreased LNCaP efferocytosis by M2 HMDMs, establishing the role of MerTK in prostate cancer efferocytosis. In the prostate cancer mouse model hi-myc, Mertk KO increased anti-tumor immune infiltrate including CD8 T cells. These findings support targeting MerTK-mediated efferocytosis as a novel therapy for prostate cancer.
Topics: Animals; Mice; Male; Humans; c-Mer Tyrosine Kinase; Prostatic Neoplasms; Phagocytosis; Macrophages; Prostate; Tumor Microenvironment
PubMed: 37644281
DOI: 10.1007/s12032-023-02153-z -
Research and Practice in Thrombosis and... Oct 2023Flow cytometry is an important technique for understanding multiple aspects of blood platelet biology. Despite the widespread use of the platform for assessing platelet...
BACKGROUND
Flow cytometry is an important technique for understanding multiple aspects of blood platelet biology. Despite the widespread use of the platform for assessing platelet function, the optimization and careful consideration of preanalytical conditions, sample processing techniques, and data analysis strategies should be regularly assessed. When set up and designed with optimal conditions, it can ensure the acquisition of robust and reproducible flow cytometry data. However, these parameters are rarely described despite their importance.
OBJECTIVES
We aimed to characterize the effects of several preanalytical variables on the analysis of blood platelets by multiparameter fluorescent flow cytometry.
METHODS
We assessed anticoagulant choice, sample material, sample processing, and storage times on 4 distinct and commonly used markers of platelet activation, including fibrinogen binding, expression of CD62P and CD42b, and phosphatidylserine exposure.
RESULTS
The use of suboptimal conditions led to increases in basal platelet activity and reduced sensitivities to stimulation; however, the use of optimal conditions protected the platelets from artifactual stimulation and preserved basal activity and sensitivity to activation.
CONCLUSION
The optimal preanalytical conditions identified here for the measurement of platelet phenotype by flow cytometry suggest a framework for future development of multiparameter platelet assays for high-quality data sets and advanced analysis.
PubMed: 37854456
DOI: 10.1016/j.rpth.2023.102205 -
The Journal of Cell Biology Feb 2024Cancer cells harness lipid metabolism to promote their own survival. We screened 47 cancer cell lines for survival dependency on phosphatidylserine (PS) synthesis using...
Cancer cells harness lipid metabolism to promote their own survival. We screened 47 cancer cell lines for survival dependency on phosphatidylserine (PS) synthesis using a PS synthase 1 (PTDSS1) inhibitor and found that B cell lymphoma is highly dependent on PS. Inhibition of PTDSS1 in B cell lymphoma cells caused a reduction of PS and phosphatidylethanolamine levels and an increase of phosphoinositide levels. The resulting imbalance of the membrane phospholipidome lowered the activation threshold for B cell receptor (BCR), a B cell-specific survival mechanism. BCR hyperactivation led to aberrant elevation of downstream Ca2+ signaling and subsequent apoptotic cell death. In a mouse xenograft model, PTDSS1 inhibition efficiently suppressed tumor growth and prolonged survival. Our findings suggest that PS synthesis may be a critical vulnerability of malignant B cell lymphomas that can be targeted pharmacologically.
Topics: Animals; Humans; Mice; Apoptosis; Lymphoma, B-Cell; Phosphatidylserines; Receptors, Antigen, B-Cell; Signal Transduction; Phosphatidylinositols; Nitrogenous Group Transferases
PubMed: 38048228
DOI: 10.1083/jcb.202212074 -
Developmental Cell Oct 2023Elucidating the mechanism(s) modulating appropriate tissue size is a critical biological issue. Pancreatic β cells increase during pregnancy via cellular proliferation,...
Elucidating the mechanism(s) modulating appropriate tissue size is a critical biological issue. Pancreatic β cells increase during pregnancy via cellular proliferation, but how β cells promptly decrease to the original amount after parturition remains unclear. Herein, we demonstrate the role and mechanism of macrophage accumulation in this process. In the final stage of pregnancy, HTR1D signaling upregulates murine β cell CXCL10, thereby promoting macrophage accumulation in pancreatic islets via the CXCL10-CXCR3 axis. Blocking this mechanism by administering an HTR1D antagonist or the CXCR3 antibody and depleting islet macrophages inhibited postpartum β cell mass reduction. β cells engulfed by macrophages increased in postpartum islets, but Annexin V administration suppressed this engulfment and the postpartum β cell mass reduction, indicating the accumulated macrophages to phagocytose β cells. This mechanism contributes to both maintenance of appropriate β cell mass and glucose homeostasis promptly adapting to reduced systemic insulin demand after parturition.
Topics: Pregnancy; Female; Mice; Animals; Insulin-Secreting Cells; Parturition; Islets of Langerhans; Insulin; Macrophages; Phagocytosis
PubMed: 37716356
DOI: 10.1016/j.devcel.2023.08.002 -
Molecules (Basel, Switzerland) Dec 2023Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In...
BACKGROUND
Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In particular, chemotherapy-related anemia is a major obstacle in cancer therapy, which may be aggravated by hemolysis and eryptosis. This work presents a toxicity assessment of RA in human RBCs and explores associated molecular mechanisms.
METHODS
RBCs isolated from healthy donors were treated with anticancer concentrations of RA (10-800 μM) for 24 h at 37 °C, and hemolysis and related markers were photometrically measured. Flow cytometry was used to detect canonical markers of eryptosis, including phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca by Fluo4/AM, cell size by FSC, and oxidative stress by HDCFDA. Ions and pH were assessed by an ion-selective electrode, while B was detected by chemiluminescence.
RESULTS
RA elicited concentration-dependent hemolysis with AST and LDH release but rescued the cells from hypotonic lysis at sub-hemolytic concentrations. RA also significantly increased annexin-V-positive cells, which was ameliorated by extracellular Ca removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B content and a decrease in FSC and extracellular pH with KCl efflux were noted upon RA treatment. Hemolysis was augmented by blocking KCl efflux and was blunted by ATP, SB203580, staurosporin, D4476, isosmotic urea, and PEG 8000.
CONCLUSIONS
RA stimulates Ca-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca accumulation, loss of ionic regulation, and cell shrinkage. These toxic effects were mediated through energy deprivation, p38 MAPK, protein kinase C, and casein kinase 1α.
Topics: Humans; Calcium; Rosmarinic Acid; Reactive Oxygen Species; Eryptosis; Hemolysis; p38 Mitogen-Activated Protein Kinases; Erythrocytes; Annexins; Phosphatidylserines
PubMed: 38138543
DOI: 10.3390/molecules28248053 -
Biomolecules Jul 2023Activated platelets are involved in blood coagulation by exposing phosphatidylserine (PS), which serves as a substrate for assembling coagulation complexes. Platelets...
Activated platelets are involved in blood coagulation by exposing phosphatidylserine (PS), which serves as a substrate for assembling coagulation complexes. Platelets accelerate fibrin formation and thrombin generation, two final reactions of the coagulation cascade. We investigated the effects of antiplatelet drugs on platelet impact in these reactions and platelet ability to expose PS. Washed human platelets were incubated with acetylsalicylic acid (ASA), ticagrelor, ASA in combination with ticagrelor, ruciromab (glycoprotein IIb-IIIa antagonist), or prostaglandin E1 (PGE1). Platelets were not activated or activated by collagen and sedimented in multiwell plates, and plasma was added after supernatant removal. Fibrin formation (clotting) was monitored in a recalcification assay by light absorbance and thrombin generation in a fluorogenic test. PS exposure was assessed by annexin V staining using flow cytometry. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, prolonged the lag phase and decreased the maximum rate of plasma clotting and decreased the peak and maximum rate of thrombin generation. Inhibition was observed when platelets were not treated with exogenous agonists (activation by endogenous thrombin) and pretreated with collagen. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, decreased PS exposure on washed platelets activated by thrombin and by thrombin + collagen. PS exposure on activated platelets in whole blood was lower in patients with acute coronary syndrome receiving ticagrelor + ASA in comparison with donors free of medications. These results indicate that antiplatelet drugs are able to suppress platelet coagulation activity not only in vitro but also after administration to patients.
Topics: Humans; Platelet Aggregation Inhibitors; Blood Platelets; Ticagrelor; Thrombin; Alprostadil; Blood Coagulation; Aspirin; Fibrin; Collagen
PubMed: 37509160
DOI: 10.3390/biom13071124 -
BioRxiv : the Preprint Server For... Sep 2023Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N) and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we find the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, lipid binding is shown to occur in the N protein C-terminal domain, which is supported by extensive analysis. Anionic lipid binding occurs for both the free and N oligomeric forms suggesting N can associate with membranes in the nucleocapsid form. Herein we present a lipid-dependent model based on , cellular and data for the recruitment of N to M assembly sites in the lifecycle of SARS-CoV-2.
PubMed: 37745364
DOI: 10.1101/2023.09.15.557899 -
Advanced Science (Weinheim,... May 2024Inflammation-responsive hydrogels loaded with therapeutic factors are effective biomaterials for bone tissue engineering and regenerative medicine. In this study, a...
Inflammation-responsive hydrogels loaded with therapeutic factors are effective biomaterials for bone tissue engineering and regenerative medicine. In this study, a matrix metalloproteinase (MMP)-responsive injectable hydrogel is constructed by integrating an MMP-cleavable peptide (pp) into a covalent tetra-armed poly-(ethylene glycol) (PEG) network for precise drug release upon inflammation stimulation. To establish a pro-regenerative environment, phosphatidylserine (PS) is encapsulated into a scaffold to form the PEG-pp-PS network, which could be triggered by MMP to release a large amount of PS during the early stage of inflammation and retain drug release persistently until the later stage of bone repair. The hydrogel is found to be mechanically and biologically adaptable to the complex bone defect area. In vivo and in vitro studies further demonstrated the ability of PEG-pp-PS to transform macrophages into the anti-inflammatory M2 phenotype and promote osteogenic differentiation, thus, resulting in new bone regeneration. Therefore, this study provides a facile, safe, and promising cell-free strategy on simultaneous immunoregulation and osteoinduction in bone engineering.
Topics: Bone Regeneration; Hydrogels; Animals; Phosphatidylserines; Immunomodulation; Matrix Metalloproteinases; Tissue Engineering; Mice; Osteogenesis; Polyethylene Glycols; Disease Models, Animal; Tissue Scaffolds; Biocompatible Materials; Models, Animal
PubMed: 38460178
DOI: 10.1002/advs.202306924 -
Cell Reports Apr 2024Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4 large peritoneal...
Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4 large peritoneal macrophages efficiently capture and cross-present tumor-associated antigens at early stages of peritoneal infiltration by ovarian cancer cells. The phosphatidylserine (PS) receptor TIM4 promotes maximal uptake of dead cells or PS-coated artificial targets and triggers inflammatory and metabolic gene programs in combination with cytoskeletal remodeling and upregulation of transcriptional signatures related to antigen processing. At the cellular level, TIM4-mediated engulfment induces nucleation of F-actin around nascent phagosomes, delaying the recruitment of vacuolar ATPase, acidification, and cargo degradation. In vivo, TIM4 deletion blunts induction of early anti-tumoral effector CD8 T cells and accelerates the progression of ovarian tumors. We conclude that TIM4-mediated uptake drives the formation of specialized phagosomes that prolong the integrity of ingested antigens and facilitate cross-presentation, contributing to immune surveillance of the peritoneum.
Topics: Animals; Macrophages, Peritoneal; Female; Mice; Carcinogenesis; Humans; Antigens, Neoplasm; Ovarian Neoplasms; Membrane Proteins; Mice, Inbred C57BL; Cross-Priming; Cell Line, Tumor; Phagosomes; Antigen Presentation; CD8-Positive T-Lymphocytes; Actins
PubMed: 38607919
DOI: 10.1016/j.celrep.2024.114096 -
The Journal of Experimental Medicine Mar 2024The intestinal epithelium is the first line of defense against enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic...
The intestinal epithelium is the first line of defense against enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal intestine. Instead, here, we describe non-professional efferocytosis of Salmonella-infected enterocytes by neighboring epithelial cells in the neonatal intestine. Intestinal epithelial stem cell organoid cocultures of neonatal and adult cell monolayers with damaged enterocytes replicated this observation, confirmed the age-dependent ability of intestinal epithelial cells for efferocytosis, and identified the involvement of the "eat-me" signals and adaptors phosphatidylserine and C1q as well as the "eat-me" receptors integrin-αv (CD51) and CD36 in cellular uptake. Consistent with this, massive epithelial cell membrane protrusions and CD36 accumulation at the contact site with apoptotic cells were observed in the infected neonatal host in vivo. Efferocytosis of infected small intestinal enterocytes by neighboring epithelial cells may represent a previously unrecognized mechanism of neonatal antimicrobial host defense to maintain barrier integrity.
Topics: Efferocytosis; Intestines; Epithelial Cells; Intestinal Mucosa; Salmonella
PubMed: 38305765
DOI: 10.1084/jem.20231237