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Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies,the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case,the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity. View Publication

High-grade serous ovarian cancer remains a poorly understood disease with a high mortality rate. Although most patients respond to cytotoxic therapies,a majority will experience recurrence. This may be due to a minority of drug-resistant cancer stem-like cells (CSC) that survive chemotherapy and are capable of repopulating heterogeneous tumors. It remains unclear how CSCs are supported in the tumor microenvironment (TME) particularly during chemotherapy exposure. Tumor-associated macrophages (TAM) make up half of the immune population of the ovarian TME and are known to support CSCs and contribute to cancer progression. TAMs are plastic cells that alter their phenotype in response to environmental stimuli and thus may influence CSC maintenance during chemotherapy. Given the plasticity of TAMs,we studied the effects of carboplatin on macrophage phenotypes using both THP1- and peripheral blood mononuclear cell (PBMC)–derived macrophages and whether this supports CSCs and ovarian cancer progression following treatment. We found that carboplatin exposure induces an M1-like proinflammatory phenotype that promotes SOX2 expression,spheroid formation,and CD117 + ovarian CSCs,and that macrophage-secreted CCL2/MCP-1 is at least partially responsible for this effect. Depletion of TAMs during carboplatin exposure results in fewer CSCs and prolonged survival in a xenograft model of ovarian cancer. This study supports a role for platinum-based chemotherapies in promoting a transient proinflammatory M1-like TAM that enriches for CSCs during treatment. Improving our understanding of TME responses to cytotoxic drugs and identifying novel mechanisms of CSC maintenance will enable the development of better therapeutic strategies for high-grade serous ovarian cancer. Significance: We show that chemotherapy enhances proinflammatory macrophage phenotypes that correlate with ovarian cancer progression. Given that macrophages are the most prominent immune cell within these tumors,this work provides the foundation for future translational studies targeting specific macrophage populations during chemotherapy,a promising approach to prevent relapse in ovarian cancer. View Publication

There is potential interest for combining allogeneic hematopoietic cell transplantation (HCT),and particularly allogeneic HCT with a nonmyeloablative regimen,to the tyrosine kinase inhibitor imatinib (Glivec; Novartis,Basel,Switzerland,http://www.novartis.com) in order to maximize anti-leukemic activity against Philadelphia chromosome-positive leukemias. However,because imatinib inhibits c-kit,the stem cell factor receptor,it could interfere with bone marrow engraftment. In this study,we examined the impact of imatinib on normal progenitor cell function. Imatinib decreased the colony-forming capacity of mobilized peripheral blood human CD133(+) cells but not that of long-term culture-initiating cells. Imatinib also decreased the proliferation of cytokine-stimulated CD133(+) cells but did not induce apoptosis of these cells. Expression of very late antigen (VLA)-4,VLA-5,and CXCR4 of CD133(+) cells was not modified by imatinib,but imatinib decreased the ability of CD133(+) cells to migrate. Finally,imatinib did not decrease engraftment of CD133(+) cells into irradiated nonobese diabetic/severe combined immunodeficient/beta2m(null) mice conditioned with 3 or 1 Gy total body irradiation. In summary,our results suggest that,despite inhibition of hematopoietic progenitor cell growth in vitro,imatinib does not interfere with hematopoietic stem cell engraftment. View Publication

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however,unipotent neutrophil progenitors are not well defined. Here,we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly,we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models,including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases,including cancer. View Publication

Both anti-tumoral and pro-tumoral effects of mesenchymal stem cells (MSCs) in preclinical treatment of ovarian cancer have been controversially demonstrated. In this study,we profiled the phenotypes of mouse compact bone-derived MSCs (CB-MSCs) and bone marrow-derived MSCs (BM-MSCs) and found that CB-MSCs expressed lower CD90 compared to BM-MSCs. We examined gene expression of immune regulating cytokines of CB-MSCs in 2D and 3D culture and under stimulation with TLR4 agonist LPS or immune activator VIC-008. Our data showed that when CB-MSCs were cultured in simulated in vivo 3D condition,CD90 expression was further decreased. Moreover,gene expressions of immune activating cytokines IL-12,IL-21,IFNgamma and a pro-inflammatory cytokine CXCL10 in CB-MSCs were increased in 3D culture whereas gene expression of anti-inflammatory cytokines IL-10 and CCL5 were downregulated. Stimulation of CB-MSCs by LPS or VIC-008 presented similar profile of the cytokine gene expressions to that in 3D culture which might benefit the anti-tumor efficacy of CD90low MSCs. The anti-tumor effects of CD90low CB-MSCs alone or in combination with VIC-008 were evaluated in a syngeneic orthotopic mouse model of ovarian cancer. Treatment that combines CB-MSCs and VIC-008 significantly decreased tumor growth and prolonged mouse survival. This was associated with the increase of activated anti-tumoral CD4+ and CD8+ T cells and the decrease of Treg cells in the tumor microenvironment. Taken together,our study demonstrates the synergistic anti-tumoral efficacy by application of CB-MSCs combined with immune activator VIC-008 and provides new insight into CD90low MSCs as a new anti-tumor arsenal. View Publication

BackgroundThe epoxyeicosatrienoic acids (EETs) are derivatives of the arachidonic acid metabolism with anti-inflammatory activities. However,their efficacy is limited due to the rapid hydrolysis by soluble epoxide hydrolase (sEH). Inhibition of sEH has been shown to stabilize the EETs and reduce neuroinflammation in A? mouse models of Alzheimer’s disease (AD). However,the role of the sEH-EET signaling pathway in other CNS cell types and neurodegenerative conditions are less understood.MethodsHere we investigated the mechanisms and functional role of the sEH-EET axis in tauopathy by treating PS19 mice with a small molecule sEH inhibitor TPPU and by crossing the PS19 mice with Ephx2 (gene encoding sEH) knockout mice. This was followed by single-nucleus RNA-sequencing (snRNA-seq),biochemical and immunohistochemical analysis,and behavioral assessments. Additionally,we examined the effects of the sEH-EET pathway in primary microglia cultures and human induced pluripotent stem cell (iPSC)-derived neurons exhibiting seeding-induced Tau inclusions.ResultssEH inhibition improved cognitive function,rescued neuronal cell loss,and reduced Tau pathology and microglial reactivity. snRNA-seq revealed that TPPU treatment upregulated genes involved in actin cytoskeleton and excitatory synaptic pathways. Treatment of human iPSC-derived neurons with TPPU enhanced synaptic density without affecting Tau accumulation,suggesting a cell-autonomous neuroprotective effect of sEH blockade. Furthermore,sEH inhibition reversed disease-associated and interferon-responsive microglial states in PS19 mice,while EET supplementation promoted Tau phagocytosis and clearance in primary microglia cultures.ConclusionThese findings demonstrate that sEH blockade or EET augmentation confers therapeutic benefit in neurodegenerative tauopathies by simultaneously targeting neuronal and microglial pathways.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00844-x. View Publication

The aorta-gonad-mesonephros region plays an important role in hematopoietic stem cell (HSC) development during mouse embryogenesis. The vascular endothelial cadherin�?� CD45�?� (VE-cad�?�CD45�?�) population contains the major type of immature pre-HSCs capable of developing into long-term repopulating definitive HSCs. In this study,we developed a new coaggregation culture system,which supports maturation of a novel population of CD45-negative (VE-cad�?�CD45�?�CD41�?�) pre-HSCs into definitive HSCs. The appearance of these pre-HSCs precedes development of the VE-cad�?�CD45�?� pre-HSCs (termed here type I and type II pre-HSCs,respectively),thus establishing a hierarchical directionality in the developing HSC lineage. By labeling the luminal surface of the dorsal aorta,we show that both type I and type II pre-HSCs are distributed broadly within the endothelial and subendothelial aortic layers,in contrast to mature definitive HSCs which localize to the aortic endothelial layer. In agreement with expression of CD41 in pre-HSCs,in vivo CD41-Cre-mediated genetic tagging occurs in embryonic pre-HSCs and persists in all lymphomyeloid lineages of the adult animal. View Publication

Regulatory T (Treg)-based cell therapy holds promise for autoimmune and inflammatory diseases,yet challenges remain regarding the functional stability and persistence of transferred Tregs. Here we engineer Tregs to express a partial agonist form of IL-2 (IL-2pa) to enhance persistence while avoiding toxicity from excessive signaling. Mouse Tregs expressing wild-type IL-2 (Tregs-IL2wt) have only a transient growth advantage,limited by toxicity from likely excessive signaling. By contrast,mouse Tregs-IL2pa exhibit sustained expansion,long-term survival in immunocompetent mice for over a year,and bystander expansion of endogenous Tregs. Tregs-IL2pa maintain a stable activated phenotype,Treg-specific demethylation,and a diverse TCR repertoire. In vivo,prophylactic transfer of Tregs-IL2pa ameliorates multi-organ autoimmunity in a Treg depletion-induced mouse autoimmune model. Lastly,compared with control Treg,human Tregs-IL2pa show enhanced survival in the IL-2-depleted environment of immune-deficient mice and improved control of xenogeneic graft-versus-host disease. Our results thus show that IL-2pa self-sufficiency enhances the stability,durability and efficacy of Treg therapies in preclinical settings. Subject terms: Cell delivery,Regulatory T cells,Autoimmune diseases,Interleukins View Publication

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified,but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved,we performed Northern analyses on RNA extracted from human marrow LTC adherent layers,or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta),IL-6,and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF,GM-CSF,and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast,transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC,and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures,respectively. Together,these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC,with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF,GM-CSF,and IL-6. Given the similarities between the LTC system and the marrow microenvironment,it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells. View Publication

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The combination of biomarkers is crucial for AD diagnosis. The triggering receptor expressed on myeloid cells 2 (TREM2),a receptor expressed on microglia,is important in AD pathogenesis. Impairment of TREM2 function aggravates the toxic effects of amyloid plaques,and its activation has been shown to reduce Aβ burden and memory deficits. Increased levels of soluble TREM2 (sTREM2) in blood and cerebrospinal fluid is associated with AD. Therefore,TREM2 could serve as a non-invasive biomarker for AD. In this study,we developed a preclinical immunoassay to detect TREM2 for AD diagnosis. Highly sensitive and specific TREM2 antibodies were produced using the hybridoma technique. The three optimized immunoassays exhibited lower limit of quantitation (LLOQ) of 0.474,0.807,and 0.415 ng/mL,respectively. These preclinical immunoassays showed high sensitivity and specificity. The sandwich enzyme-linked immunosorbent assay (ELISA) could potentially be used for AD diagnosis. View Publication

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity,we developed a fluorescent substrate for ALDH,termed BODIPY aminoacetaldehyde (BAAA),and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells,40-90% pure for CD34(+)CD38(lo/-) cells,and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together,these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well. View Publication

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins,the widely expressed basic helix-loop-helix transcription factors,contribute to HSC and MPP activity,but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches,we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However,long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover,E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism,and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together,these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism,and demonstrate that E47 is not required for short-term myeloid differentiation. View Publication