搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Acute myeloid leukemia (AML) is primarily driven by leukemic stem cells (LSCs),the main cause of relapse and therapy resistance. Here,we discover that LSCs are predominantly small and mechanically soft. These mechanical properties enable their selective isolation using microfluidic chips. Single-cell RNA-sequencing of primary human AML bone marrow identifies enrichment of LSCs within the FSClow ALDH1A1+ subpopulation,which exhibits long-term stemness in functional assays. Notably,inhibiting ALDH1A1 in these cells promotes F-actin polymerization and increases cellular stiffness,reducing their stemness while enhancing their susceptibility to natural killer (NK) cell-mediated cytotoxicity. In AML patient-derived xenograft models,the combination of ALDH1A1 inhibition with NK cell therapy markedly suppresses leukemia progression. These findings suggest that targeting the mechanical properties of LSC offers a promising strategy to overcome AML treatment resistance,providing insights into stem cell mechanobiology and paving the way for combining targeted therapies with immunotherapy to improve clinical outcomes. Leukemic stem cells (LSCs) drive relapse and therapy resistance in acute myeloid leukemia (AML). Here,the authors show that increasing the stiffness of LSCs reduces their stemness and enhances their susceptibility to natural killer cell-mediated immunotherapy in AML. View Publication

YAP,a key effector of Hippo pathway,is activated by its translocation from cytoplasm to nucleus to regulate gene expression and promote tumorigenesis. Although the mechanism by which YAP is suppressed in cytoplasm has been well-studied,how the activated YAP is sequestered in the nucleus remains unknown. Here,we demonstrate that YAP is a nucleocytoplasmic shuttling protein and its nuclear export is controlled by SET1A-mediated mono-methylation of YAP at K342,which disrupts the binding of YAP to CRM1. YAP mimetic methylation knockin mice are more susceptible to colorectal tumorigenesis. Clinically,YAP K342 methylation is reversely correlated with cancer survival. Collectively,our study identifies SET1A-mediated mono-methylation at K342 as an essential regulatory mechanism for regulating YAP activity and tumorigenesis. View Publication

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that,in human,the first lineage restriction events occur within the CD19-CD34+CD38-CD45RA-CD49f+CD90+ (49f+) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution,we observe a continuous but polarised organisation of the 49f+ compartment,where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9AhiCD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics,whereas CLEC9AloCD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state,distinct from lymphoid-primed multipotent progenitors,representing the earliest entry point into lymphoid commitment. View Publication

In life sciences,the material properties of suspended cells have attained significance close to that of fluorescent markers but with the advantage of label-free and unbiased sample characterization. Until recently,cell rheological measurements were either limited by acquisition throughput,excessive post processing,or low-throughput real-time analysis. Real-time deformability cytometry expanded the application of mechanical cell assays to fast on-the-fly phenotyping of large sample sizes,but has been restricted to single material parameters as the Young's modulus. Here,we introduce dynamic real-time deformability cytometry for comprehensive cell rheological measurements at up to 100 cells per second. Utilizing Fourier decomposition,our microfluidic method is able to disentangle cell response to complex hydrodynamic stress distributions and to determine viscoelastic parameters independent of cell shape. We demonstrate the application of our technology for peripheral blood cells in whole blood samples including the discrimination of B- and CD4+ T-lymphocytes by cell rheological properties. View Publication

Tissue inhibitor matrix metalloproteinase-1 (TIMP1) is one of four identified members of the TIMP family. We evaluated the role of TIMP1 in gastric cancer using human and mouse tissues along with gastric organoids and in vitro cell models. Using quantitative real-time RT-PCR,we detected significant overexpression of TIMP1 in the human gastric cancer samples,as compared to normal stomach samples (P {\textless} 0.01). We also detected overexpression of Timp1 in neoplastic gastric lesions of the Tff1-knockout (KO) mice,as compared to normal stomach tissues. Reconstitution of TFF1 in human gastric cancer cell lines led to a significant decrease in the mRNA expression level of TIMP1 (P {\textless} 0.05). In vitro analysis demonstrated that TIMP1 mRNA expression is induced by TNF-alpha and activation of NF-kappaB whereas inhibition of NF-kappaB using BAY11-7082 led to inhibition of NF-kappaB and downregulation of TIMP1. Western blot analysis confirmed the decrease in TIMP1 protein level following reconstitution of TFF1. By using immunofluorescence,we showed nuclear localization of NF-kappaB and expression of TIMP1 in gastric organoids established from the Tff1-KO stomach where reconstitution of Tff1 using recombinant protein led to a notable reduction in the expression of both NF-kappaB and TIMP1. Using EDU assay,as a measure of proliferating cells,we found that TIMP1 promotes cellular proliferation whereas TFF1 reconstitution leads to a significant decrease in cellular proliferation (P {\textless} 0.05). In summary,our findings demonstrate overexpression of TIMP1 in mouse and human gastric cancers through NF-kB-dependent mechanism. We also show that TFF1 suppresses NF-kappaB and inhibits TIMP1-mediated proliferative potential in gastric cancer. View Publication

Intratumoral (IT) STING activation results in tumor regression in preclinical models,yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here,clinical candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a T cell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to high-dose tumor ablative regimens that result in systemic S100 distribution,low-dose immunogenic regimens induce local activation of tumor-specific CD8+ effector T cells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific T cell activation,and in the context of optimized T cell responses,TNFalpha is dispensable for tumor control. In a poorly immunogenic model,S100 combined with CPIs generates a survival benefit and durable protection. These results provide fundamental mechanistic insights into STING-induced anti-tumor immunity. View Publication

BACKGROUND: Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However,few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-beta1 and calcitriol. DESIGN: In vitro study employing two models of normal human osteoblasts: human bone marrow stromal cells [hMS/(OB)] containing osteoprogenitor cells and trabecular bone osteoblasts (hOB),which are mature osteoblasts. A reverse-transcriptase-polymerase-chain-reaction assay was employed to measure steady state mRNA levels of TGF-beta(s) isoforms and receptors. Effects of short-term treatment of TGF-beta1 on osteoblast proliferation and differentiation markers were assessed. The effect of cotreatment of calcitriol (10-8 M) and TGF-beta1 on osteoblast differentiation was also determined. RESULTS: Both hMS(OB) and hOB cells expressed mRNA transcripts of TGF-beta1,TGF-beta2,TGF-beta 3,TGF-beta type I and type II receptors. TGF-beta 1 stimulated osteoblast proliferation in hMS(OB) and in hOB cultures. In hOB cultures,TGF-beta1 stimulated AP production and cotreatment with calcitriol induced a synergistic increase in AP levels to 250 +/- 61% of calcitriol-treated controls. Effects of TGF-beta1 and calcitriol were less pronounced in hMS(OB) cultures. TGF-beta1 inhibited collagen type I production in hMS(OB) cells and these effects were abolished in presence of calcitriol. In presence of calcitriol,TGF-beta1 increased collagen type I production in hOB cells. In both hOB and hMS(OB) cultures,TGF-beta1 inhibited osteocalcin production. CONCLUSIONS: TGF-beta increases osteoblastic cell proliferation irrespective of the differentiation state. In presence of calcitriol,it initiates osteoblast cell differentiation and matrix formation. As TGF-beta inhibits osteocalcin production,other factors are necessary for inducing terminal differentiation of osteoblasts. The observed effects of TGF-beta on human osteoblasts in vitro may represent important regulatory steps in controlling osteoblast cell proliferation and differentiation in vivo. View Publication

The molecular mechanisms by which tumor cells metastasize to bone are likely to involve invasion,cell adhesion to bone,and the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are,therefore,used in the treatment of patients with osteolytic metastases. However,an added beneficial effect of BPs may be direct antitumor activity. We previously reported that BPs inhibit breast and prostate carcinoma cell adhesion to bone (Boissier et al.,Cancer Res.,57: 3890-3894,1997). Here,we provided evidence that BP pretreatment of breast and prostate carcinoma cells inhibited tumor cell invasion in a dose-dependent manner. The order of potency for four BPs in inhibiting tumor cell invasion was: zoledronate textgreater ibandronate textgreater NE-10244 (active pyridinium analogue of risedronate) textgreater clodronate. In addition,NE-58051 (the inactive pyridylpropylidene analogue of risedronate) had no inhibitory effect,whereas NE-10790 (a phosphonocarboxylate analogue of risedronate in which one of the phosphonate groups is substituted by a carboxyl group) inhibited tumor cell invasion to an extent similar to that observed with NE-10244,indicating that the inhibitory activity of BPs on tumor cells involved the R2 chain of the molecule. BPs did not induce apoptosis in tumor cells,nor did they inhibit tumor cell migration at concentrations that did inhibit tumor cell invasion. However,although BPs did not interfere with the production of matrix metalloproteinases (MMPs) by tumor cells,they inhibited their proteolytic activity. The inhibitory effect of BPs on MMP activity was completely reversed in the presence of an excess of zinc. In addition,NE-10790 did not inhibit MMP activity,suggesting that phosphonate groups of BPs are responsible for the chelation of zinc and the subsequent inhibition of MMP activity. In conclusion,our results provide evidence for a direct cellular effect of BPs in preventing tumor cell invasion and an inhibitory effect of BPs on the proteolytic activity of MMPs through zinc chelation. These results suggest,therefore,that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone. View Publication

There is no known treatment for fatty liver,a ubiquitous cause of chronic liver disease. However,because it is associated with hyperinsulinemia and insulin-resistance,insulin-sensitizing agents might be beneficial. To evaluate this possibility,insulin-resistant ob/ob mice with fatty livers were treated with metformin,an agent that improves hepatic insulin-resistance. Metformin improved fatty liver disease,reversing hepatomegaly,steatosis and aminotransferase abnormalities. The therapeutic mechanism likely involves inhibited hepatic expression of tumor necrosis factor (TNF) alpha and TNF-inducible factors that promote hepatic lipid accumulation and ATP depletion. These findings suggest a mechanism of action for metformin and identify novel therapeutic targets in insulin-resistant states. View Publication

Two conformationally restricted analogues of (-)-indolactam-V (1) (cis and trans amides) were examined for their binding selectivity to the synthetic C1 peptides of all protein kinase C (PKC) isozymes. Although the binding constants of the cis amide-restricted analogue (2) were equal to those of 1,the trans amide-restricted analogue (3) bound significantly only to the novel PKC (delta,epsilon,eta,theta) isozymes. View Publication