Reemergence of a Big Brown Bat Lyssavirus rabies Variant in Striped Skunks in Flagstaff, Arizona, USA, 2021-2023.

Background: Throughout the Americas, Lyssavirus rabies (RV) perpetuates as multiple variants among bat and mesocarnivore species. Interspecific RV spillover occurs on occasion, but clusters and viral host shifts are rare. The spillover and host shift of a big brown bat (Eptesicus fuscus) RV variant Ef-W1 into mesocarnivores was reported previously on several occasions during 2001-2009 in Flagstaff, Arizona, USA, and controlled through rabies vaccination of target wildlife. During autumn 2021, a new cluster of Ef-W1 RV cases infecting striped skunks (Mephitis mephitis) was detected from United States Department of Agriculture enhanced rabies surveillance in Flagstaff. The number of Ef-W1 RV spillover cases within a short timeframe suggested the potential for transmission between skunks and an emerging host shift. Materials and Methods: Whole and partial RV genomic sequencing was performed to evaluate the phylogenetic relationships of the 2021-2023 Ef-W1 cases infecting striped skunks with earlier outbreaks. Additionally, real-time reverse-transcriptase PCR (rtRT-PCR) was used to opportunistically compare viral RNA loads in brain and salivary gland tissues of naturally infected skunks. Results: Genomic RV sequencing revealed that the origin of the 2021-2023 epizootic of Ef-W1 RV was distinct from the multiple outbreaks detected from 2001-2009. Naturally infected skunks with the Ef-W1 RV showed greater viral RNA loads in the brain, but equivalent viral RNA loads in the mandibular salivary glands, compared to an opportunistic sample of skunks naturally infected with a South-Central skunk RV from northern Colorado, USA. Conclusion: Considering a high risk for onward transmission and spread of the Ef-W1 RV in Flagstaff, public outreach, enhanced rabies surveillance, and control efforts, focused on education, sample characterization, and vaccination, have been ongoing since 2021 to mitigate and prevent the spread and establishment of Ef-W1 RV in mesocarnivores.

Platelets retain function and can be stored following disruption of human leucocyte antigens.

BACKGROUND AND OBJECTIVES: Antibodies to human leucocyte antigen (HLA) Class-I antigens can lead to refractoriness to platelet transfusion. Although this can be overcome by transfusion of HLA-compatible platelets, they are not always available. Disruption of HLA antigens on platelets by acid treatment may be a suitable alternative when no other components are available. The aim of this study was to assess the effect of HLA disruption and subsequent storage of platelet components. MATERIALS AND METHODS: Platelet components were treated with 0.9% saline or citric acid solution (pH 3.0), and then stored until expiry (Day 7). HLA and platelet glycoprotein expression, platelet viability, activation and sialylation were measured by flow cytometry. Release of soluble factors was measured by ELISA and metabolism by biochemistry analyser. Reactivity to patient anti-sera containing anti-HLA antibodies was measured using platelet immunofluorescence tests (PIFTs) and monoclonal antibody immobilization of platelet antigen (MAIPA) assays. Platelet function was measured using aggregometry and thromboelastography (TEG). RESULTS: Acid treatment reduced detection of HLA Class-I on platelets by 75%, with significant reductions in reactivity to patient anti-sera. Acid treatment reduced platelet content and viability, increased platelet activation and accelerated metabolism. Glycan cleavage was increased by acid treatment. Treatment reduced platelet activation following agonist stimulation by ADP and TRAP-6, but platelets remained functional, displaying increased aggregation response and reduced time to clot formation by TEG. CONCLUSION: Although HLA disruption had some detrimental effects, acid-treated platelets remained functional, retaining their capacity to respond to agonists and form clots, and with further development could be used to support refractory patients.

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test.

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. This test provides rapid results and can be used to detect rabies antibodies in several different scenarios. The rabies IFA test is especially useful for the quick and early detection of antibodies to evaluate the immune response in a patient who has developed rabies. Although other methods for antemortem rabies diagnosis take precedence, this test may be utilized to demonstrate recent rabies virus exposure through antibody detection. The IFA test does not provide a virus-neutralizing antibody (VNA) titer, but the pre-exposure prophylaxis (PrEP) response can be evaluated through positive or negative antibody presence. This test can be utilized in various situations and can provide results for a number of different targets. In this study, we used several paired serum samples from individuals who received PrEP and demonstrated their rabies antibody presence over time using the IFA test.

Coinfection of Canine Distemper Virus and Rabies Virus in Wildlife Samples Submitted for Routine Rabies Testing.

Canine distemper virus (also known as Canine morbillivirus), the etiologic agent of canine distemper, is a highly contagious pathogen causing a multisystemic infection in carnivores globally. Canine distemper may be clinically indistinguishable from rabies, and outbreaks of either disease are major concerns. In the US, both diseases are endemic and managed by parenteral vaccination in domestic animals. In wildlife, oral vaccination and trap-vaccinate-release programs are available for rabies prevention, but no such strategies exist for canine distemper. We evaluated the prevalence at which canine distemper virus occurred concurrently in animals infected with rabies virus. Real-time quantitative reverse transcriptase PCR (qRT-PCR) was performed on specimens previously diagnosed with rabies during 2017-19 by the New York State Rabies Laboratory. Real-time qRT-PCR detected concurrent canine distemper virus infection in 73 of 1,302 animals with rabies virus. Coinfection rates were approximately 9% in Procyon lotor, 2% in Vulpes vulpes, and 0.4% in Mephitis mephitis, with an overall prevalence of 5.6%. As comorbidities in wildlife occur, laboratory-based surveillance and confirmatory testing are critical to rapid decision making for disease prevention. Rabies virus incursions are expensive and difficult to manage, and spillover events create health risks to humans and domestic animals as well as to free-roaming wildlife.

Phylogenomic Diversity Elucidates Mechanistic Insights into Lyme Borreliae-Host Association.

Host association-the selective adaptation of pathogens to specific host species-evolves through constant interactions between host and pathogens, leaving a lot yet to be discovered on immunological mechanisms and genomic determinants. The causative agents of Lyme disease (LD) are spirochete bacteria composed of multiple species of the Borrelia burgdorferi sensu lato complex, including B. burgdorferi (Bb), the main LD pathogen in North America-a useful model for the study of mechanisms underlying host-pathogen association. Host adaptation requires pathogens' ability to evade host immune responses, such as complement, the first-line innate immune defense mechanism. We tested the hypothesis that different host-adapted phenotypes among Bb strains are linked to polymorphic loci that confer complement evasion traits in a host-specific manner. We first examined the survivability of 20 Bb strains in sera in vitro and/or bloodstream and tissues in vivo from rodent and avian LD models. Three groups of complement-dependent host-association phenotypes emerged. We analyzed complement-evasion genes, identified a priori among all strains and sequenced and compared genomes for individual strains representing each phenotype. The evolutionary history of ospC loci is correlated with host-specific complement-evasion phenotypes, while comparative genomics suggests that several gene families and loci are potentially involved in host association. This multidisciplinary work provides novel insights into the functional evolution of host-adapted phenotypes, building a foundation for further investigation of the immunological and genomic determinants of host association. IMPORTANCE Host association is the phenotype that is commonly found in many pathogens that preferential survive in particular hosts. The Lyme disease (LD)-causing agent, B. burgdorferi (Bb), is an ideal model to study host association, as Bb is mainly maintained in nature through rodent and avian hosts. A widespread yet untested concept posits that host association in Bb strains is linked to Bb functional genetic variation conferring evasion to complement, an innate defense mechanism in vertebrate sera. Here, we tested this concept by grouping 20 Bb strains into three complement-dependent host-association phenotypes based on their survivability in sera and/or bloodstream and distal tissues in rodent and avian LD models. Phylogenomic analysis of these strains further correlated several gene families and loci, including ospC, with host-specific complement-evasion phenotypes. Such multifaceted studies thus pave the road to further identify the determinants of host association, providing mechanistic insights into host-pathogen interaction.

Cellular and immunological mechanisms influence host-adapted phenotypes in a vector-borne microparasite.

Predicting pathogen emergence and spillover risk requires understanding the determinants of a pathogens' host range and the traits involved in host competence. While host competence is often considered a fixed species-specific trait, it may be variable if pathogens diversify across hosts. Balancing selection can lead to maintenance of pathogen polymorphisms (multiple-niche-polymorphism; MNP). The causative agent of Lyme disease, Borrelia burgdorferi (Bb), provides a model to study the evolution of host adaptation, as some Bb strains defined by their outer surface protein C (ospC) genotype, are widespread in white-footed mice and others are associated with non-rodent vertebrates (e.g. birds). To identify the mechanisms underlying potential strain × host adaptation, we infected American robins and white-footed mice, with three Bb strains of different ospC genotypes. Bb burdens varied by strain in a host-dependent fashion, and strain persistence in hosts largely corresponded to Bb survival at early infection stages and with transmission to larvae (i.e. fitness). Early survival phenotypes are associated with cell adhesion, complement evasion and/or inflammatory and antibody-mediated removal of Bb, suggesting directional selective pressure for host adaptation and the potential role of MNP in maintaining OspC diversity. Our findings will guide future investigations to inform eco-evolutionary models of host adaptation for microparasites.

Donor citrate reactions influence the phenotype of apheresis platelets following storage.

BACKGROUND: Platelet collection and processing methods, as well as donor attributes, can influence platelet function and quality during ex vivo storage. In this study, activation and procoagulant responses in platelets collected from donors experiencing a citrate reaction (CR) were investigated. STUDY DESIGN AND METHODS: Apheresis platelet components (n = 54) were stored in 100% autologous plasma and tested on days 1 and 5 post-collection. Platelet components were categorized into two groups according to whether the donor had experienced a CR during donation (n = 10; non-CR group, n = 44). Platelet aggregation was initiated with collagen and thrombin. Platelet phenotype was characterized by flow cytometry. Fibrinogen binding was assessed following collagen + thrombin stimulation (COATed platelets), and procoagulant activity was assessed using a procoagulant phospholipid assay (PPL). Platelet microparticle (PMP) subsets were enumerated by flow cytometry. RESULTS: Basal von Willebrand factor (VWF) binding was higher in the CR donations when compared with the non-CR group. Collagen aggregation was significantly higher in platelets from CR donations, in contrast to aggregation induced by thrombin. The proportion of phosphatidylserine (PS) positive PMP and PPL clotting time were higher in the CR group, in contrast to the number of basal PS+ platelets and COATed platelets following stimulation. CONCLUSION: Platelets donated by donors who experienced a CR during donation had higher platelet activation response and possibly a more procoagulant PMP phenotype, suggesting that this donor reaction might lead to increased platelet activation.

X-irradiation and gamma-irradiation inactivate lymphocytes in blood components.

BACKGROUND: Irradiation of selected blood components is standard practice for the prevention of transfusion-associated graft-versus-host disease (TA-GvHD). Currently, gamma-irradiation is the most widely used form of irradiation, but there is an increasing interest in X-irradiation, which is considered to be functionally equivalent and safer. However, there is a paucity of contemporary data regarding the ability of X-irradiation to inactivate lymphocytes in blood components. Therefore, the effect of gamma- and X-irradiation on lymphocyte viability and function in blood components was compared. STUDY DESIGN AND METHODS: Lymphocytes were isolated from venous blood by density gradient centrifugation, spiked into plasma/SSP+ to simulate a blood component, and either gamma- or X-irradiated. The phenotype of the isolated lymphocytes was confirmed. Lymphocyte viability was measured using a LIVE/DEAD assay, and function was assessed using mixed lymphocyte culture and CD69 expression post-phorbol-12 myristate 13-acetate (PMA) stimulation. RESULTS: Lymphocyte viability and CD69 expression following PMA stimulation were significantly reduced by both gamma-irradiation and X-irradiation in simulated blood components. Allorecognition and allostimulation were also significantly reduced by both gamma-irradiation and X-irradiation. CONCLUSION: Lymphocyte viability and function are reduced to a similar extent by gamma- and X-irradiation in simulated blood components. As such, X-irradiation is suitable for the irradiation of blood components and, in terms of lymphocyte inactivation, could be used instead of gamma-irradiation.

Feline and Canine Rabies in New York State, USA.

In New York State, domestic animals are no longer considered rabies vector species, but given their ubiquity with humans, rabies cases in dogs and cats often result in multiple individuals requiring post-exposure prophylaxis. For over a decade, the New York State rabies laboratory has variant-typed these domestic animals to aid in epidemiological investigations, determine exposures, and generate demographic data. We produced a data set that outlined vaccination status, ownership, and rabies results. Our data demonstrate that a large percentage of felines submitted for rabies testing were not vaccinated or did not have a current rabies vaccination, while canines were largely vaccinated. Despite massive vaccination campaigns, free clinics, and education, these companion animals still occasionally contract rabies. Barring translocation events, we note that rabies-positive cats and dogs in New York State have exclusively contracted a raccoon variant. While the United States has made tremendous strides in reducing its rabies burden, we hope these data will encourage responsible pet ownership including rabies vaccinations to reduce unnecessary animal mortality, long quarantines, and post-exposure prophylaxis in humans.

Heparan sulfates from bat and human lung and their binding to the spike protein of SARS-CoV-2 virus.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has resulted in a pandemic and continues to spread at an unprecedented rate around the world. Although a vaccine has recently been approved, there are currently few effective therapeutics to fight its associated disease in humans, COVID-19. SARS-CoV-2 and the related severe acute respiratory syndrome (SARS-CoV-1), and Middle East respiratory syndrome (MERS-CoV) result from zoonotic respiratory viruses that have bats as the primary host and an as yet unknown secondary host. While each of these viruses has different protein-based cell-surface receptors, each rely on the glycosaminoglycan, heparan sulfate as a co-receptor. In this study we compare, for the first time, differences and similarities in the structure of heparan sulfate in human and bat lungs. Furthermore, we show that the spike glycoprotein of COVID-19 binds 3.5 times stronger to human lung heparan sulfate than bat lung heparan sulfate.

Association of the invasive Haemaphysalis longicornis tick with vertebrate hosts, other native tick vectors, and tick-borne pathogens in New York City, USA.

Haemaphysalis longicornis, the Asian longhorned tick, is an invasive ixodid tick that has rapidly spread across the northeastern and southeastern regions of the United States since first reported in 2017. The emergence of H. longicornis presents a potential threat for livestock, wildlife, and human health as the host associations and vector potential of this invasive pest in the United States are poorly understood. Previous field data from the United States has shown that H. longicornis was not associated with natural populations of small mammals or birds, but they show a preference for medium sized mammals in laboratory experiments. Therefore, medium and large sized mammals were sampled on Staten Island, New York, United States, to determine H. longicornis host associations and vector potential for a range of human and veterinary pathogens. A total of 97 hosts were sampled and five species of tick (Amblyomma americanum, Dermacentor variabilis, H. longicornis, Ixodes scapularis, Ixodes cookei) were found feeding concurrently on these hosts. Haemaphysalis longicornis was found in the highest proportions compared with other native tick species on raccoons (55.4%), Virginia opossums (28.9%), and white-tailed deer (11.5%). Tissue, blood, and engorged larvae were tested for 17 different pathogens using a nanoscale PCR platform. Infection with five pathogens (Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia spp., Mycoplasma haemocanis, and Bartonella spp.) was detected in host samples, but no pathogens were found in any larval samples. These results suggest that although large and medium sized mammals feed large numbers of H. longicornis ticks in the environment, there is presently a low potential for H. longicornis to acquire pathogens from these wildlife hosts.

Detection of rabies viral neutralizing antibodies in the Puerto Rican Brachyphylla cavernarum.

The purpose of this study was to determine if Puerto Rican bats had previous exposure to rabies virus based on viral neutralizing antibodies. Our results demonstrate that 6.5% of the bats in this study had some exposure to rabies virus. The route of exposure is unknown but may have occurred following interaction with a rabid terrestrial animal or an unidentified bat rabies virus.

Origin of 3 Rabid Terrestrial Animals in Raccoon Rabies Virus-Free Zone, Long Island, New York, USA, 2016-2017.

During 2016-2017, three rabid terrestrial animals were discovered in the raccoon rabies virus-free zone of Long Island, New York, USA. Whole-genome sequencing and phylogenetic analyses revealed the likely origins of the viruses, enabling the rabies outbreak response (often costly and time-consuming) to be done less expensively and more efficiently.

A Phytosterolemic Mixture of Sterols Inhibits Cholesterol Synthesis, Esterification, and Low-Density Lipoprotein Receptor mRNA Abundance in HepG2 Cells.

HepG2 cells were incubated with a 16.5:1.7:1 ratio of cholesterol:sitosterol:campesterol (CSC), a ratio of the major sterols observed in the plasma of phytosterolemia patients, or with cholesterol alone in combination with [14 C]acetate for 24 h and the radioactivity incorporated into lipids determined. Cells incubated with CSC exhibited a 40% reduction in cholesterol esterification (p < 0.05) compared to cells incubated with cholesterol alone. In addition, a 17.5-fold reduction (p < 0.05) in total cholesterol (cholesterol plus cholesteryl ester) synthesis from [14 C]acetate was observed in cells incubated with CSC compared to cholesterol alone. Low-density lipoprotein receptor (LDLR) mRNA abundance was lower in cells incubated with CSC compared to cells incubated with cholesterol alone. Our results suggest that incubation of HepG2 cells with a ratio of sterols that mimic the plasma concentration seen in phytosterolemia patients reduces cholesterol esterification, total cholesterol synthesis, and inhibits LDLR mRNA abundance. We suggest that future cell and animal-based work on phytostosterolemia might employ this methodology to serve as a novel paradigm of the disease.

The role of neuraminidase 1 and 2 in glycoprotein Ibα-mediated integrin αIIbß3 activation.

Upon vascular injury, platelets adhere to von Willebrand Factor (VWF) via glycoprotein Ibα (GPIbα). GPIbα contains many glycans, capped by sialic acid. Sialic acid cleavage (desialylation) triggers clearance of platelets. Neuraminidases (NEU) are responsible for desialylation and so far, NEU1-4 have been identified. However, the role of NEU in healthy platelets is currently unknown. Aim of the study was to study the role of NEU1 and NEU2 in platelet signalling. Membrane association of platelet attached glycans, NEU1 and NEU2 was measured following activation with agonists using flow cytometry. Adhesion on fibrinogen, aggregation and fibrinogen-binding were assessed with/without the NEU-inhibitor, 2-deoxy-2-3-dide-hydro-N-acetylneuraminic acid. Cellular localisation of NEU1 and NEU2 was examined by fluorescence microscopy. Desialylation occurred following GPIbα-clustering by VWF. Basal levels of membrane NEU1 were low; glycoprotein Ibα-clustering induced a four-fold increase (n=3, P<0.05). Inhibition of αIIbß3-integrin prevented the increase in NEU1 membrane-association by ~60%. Membrane associated NEU2 increased two-fold (n=3, P<0.05) upon VWF-binding, while inhibition/removal of GPIbα reduced the majority of membrane associated NEU1 and NEU2 (n=3, P<0.05). High shear and addition of fibrinogen increased membrane NEU1 and NEU2. NEU-inhibitior prevented VWF-induced αIIbß3-integrin activation by 50% (n=3, P<0.05), however, promoted VWF-mediated agglutination, indicating a negative feedback mechanism for NEU activity. NEU1 or NEU2 were partially co-localised with mitochondria and α-granules respectively. Neither NEU1 nor NEU2 co-localised with lysosomal-associated membrane protein 1. These findings demonstrate a previously unrecognised role for NEU1 and NEU2 in GPIbα-mediated and αIIbß3-integrin signalling.

The evolution of a bat population with white-nose syndrome (WNS) reveals a shift from an epizootic to an enzootic phase.

BACKGROUND: White-nose Syndrome (WNS) is a mycosis caused by a cutaneous infection with the fungus Pseudogymnoascus destructans (Pd). It produces hibernation mortality rates of 75-98% in 4 bats: Myotis lucifugus, M. septentrionalis, M. sodalis, and Perimyotis subflavus. These high mortality rates were observed during the first several years after the arrival of P. destructans at a hibernation site. Mortality is caused by a 60% decrease in torpor bout duration, which results in a premature depletion of depot fat prior to spring. RESULTS: Little is known about the long-term effects of Pd on torpor and mortality, thus we conducted a 9-year study on M. lucifugus at 5 of the hibernation sites where Pd first appeared in North America during the winter of 2007-08. The M. lucifugus hibernating at one of these sites one year after the arrival of Pd (2008-09) had: a) a mean torpor bout duration of 7.6 d, b) no depot fat reserves by March, and c) an apparent over-winter mortality rate of 88%. The M. lucifugus hibernating at this same site 6-9 years after the arrival of Pd, in contrast, had: a) a mean torpor bout duration of 14.7 d, b) depot fat remaining in March, and c) an apparent mortality rate of 50%. The number of M. lucifugus hibernating at 2 of these sites has consistently increased since 2010 and is now more than 3.0-fold higher than the number remaining after the winter of 2008-09. CONCLUSIONS: These findings indicate that this population of M. lucifugus has evolved mechanisms to hibernate well in the presence of Pd, thus reducing over-winter mortality.

Rabies Necropsy Techniques in Large and Small Animals.

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the entire state, with the exception of New York City. The Rabies laboratory necropsies a variety of animals ranging in size from bats to bovids. Most of these specimens are animals exhibiting neurological signs, however, less than 10% actually test positive for rabies; implying trauma, lesions or other infectious agents as the cause of these symptoms. Due to the risk of aerosolizing undiagnosed infectious agents, the Rabies Laboratory does not use power tools or saws. Three necropsy techniques will be presented for animals whose skulls are impenetrable with scissors. The laboratory has implemented these techniques to decrease potential exposure to infectious agents, eliminate unnecessary manipulation of the specimen and reduce processing time. The advantages of a preferred technique opposed to another is subject to the trained individual processing the specimen.

Clarifying Indeterminate Results on the Rabies Direct Fluorescent Antibody Test Using Real-Time Reverse Transcriptase Polymerase Chain Reaction.

OBJECTIVES: Each year, rabies virus infection results in the death of more than 50 000 persons worldwide. In the United States, the Centers for Disease Control and Prevention (CDC) reported 23 human rabies cases from May 1, 2008, through October 1, 2017. Although rabies testing in the United States is highly reliable, some specimens submitted to rabies laboratories do not have adequate tissues or may be substantially decomposed. In these instances, the specimen may be considered unsatisfactory for testing or produce indeterminate results using the gold standard direct fluorescent antibody test. The objective of this study was to evaluate the number of unsatisfactory samples or samples with indeterminate results that were positive for rabies virus after additional testing using real-time reverse transcriptase polymerase chain reaction (RT-PCR). METHODS: In 2016, we retested all unsatisfactory specimens or specimens with indeterminate results using real-time RT-PCR. We further typed any sample that was real-time RT-PCR positive to identify the infecting rabies virus variant. RESULTS: Of 210 retested unsatisfactory specimens or specimens with indeterminate results, 9 (4.3%) were positive for rabies. In each case, the animal was infected with a homologous rabies virus variant. CONCLUSION: These results confirm the recommendation by CDC and state public health laboratories that indeterminate results should be considered positive and justify the prompt treatment of exposed persons through an animal that is suspected to have rabies.

The EGFR T790M Mutation Is Acquired through AICDA-Mediated Deamination of 5-Methylcytosine following TKI Treatment in Lung Cancer.

: Almost all patients with EGFR-driven lung cancer who are treated with EGFR tyrosine kinase inhibitors (TKI) develop resistance to treatment. A single base (c.2369C>T) transition mutation, EGFR T790M, is the most frequent resistance event after first-generation exposure to EGFR TKIs. Whether T790M mutation is acquired or is selected from a preexisting clone has been a matter of significant debate. In this study, we show that treatment with EGFR TKIs leads to activation of the NFκB pathway, which in turn induces expression of activation-induced cytidine deaminase (AICDA). In turn, AICDA causes deamination of 5-methylcytosine to thymine at position c.2369 to generate the T790M mutation. Pharmacologic inhibition of the NFκB pathway or knockout of AICDA decreased the frequency or prevented the development of T790M mutation, respectively. In addition, patients treated with first-line EGFR TKI displayed increased expression of AICDA and detection of the T790M mutation upon progression. These results identify the mechanism of T790M acquisition and present an opportunity to target the process to delay or prevent it. SIGNIFICANCE: These findings identify the mechanism behind acquisition of a common resistance mutation to TKI treatment in lung cancer.

Targeting Breast Cancer Stem Cell State Equilibrium through Modulation of Redox Signaling.

Although breast cancer stem cells (BCSCs) display plasticity transitioning between quiescent mesenchymal-like (M) and proliferative epithelial-like (E) states, how this plasticity is regulated by metabolic or oxidative stress remains poorly understood. Here, we show that M- and E-BCSCs rely on distinct metabolic pathways and display markedly different sensitivities to inhibitors of glycolysis and redox metabolism. Metabolic or oxidative stress generated by 2DG, H2O2, or hypoxia promotes the transition of ROSlo M-BCSCs to a ROShi E-state. This transition is reversed by N-acetylcysteine and mediated by activation of the AMPK-HIF1α axis. Moreover, E-BCSCs exhibit robust NRF2-mediated antioxidant responses, rendering them vulnerable to ROS-induced differentiation and cytotoxicity following suppression of NRF2 or downstream thioredoxin (TXN) and glutathione (GSH) antioxidant pathways. Co-inhibition of glycolysis and TXN and GSH pathways suppresses tumor growth, tumor-initiating potential, and metastasis by eliminating both M- and E-BCSCs. Exploiting metabolic vulnerabilities of distinct BCSC states provides a novel therapeutic approach targeting this critical tumor cell population.