技术资料

Acute myeloid leukemia (AML) is a heterogeneous group of aggressive bone marrow cancers arising from transformed hematopoietic stem and progenitor cells (HSPC). Therapy-related AML and MDS (t-AML/MDS) comprise a subset of AML cases occurring after exposure to alkylating chemotherapy and/or radiation and are associated with a very poor prognosis. Less is known about the pathogenesis and disease-initiating/leukemia stem cell (LSC) subpopulations of t-AML/MDS compared to their de novo counterparts. Here,we report the development of mouse models of t-AML/MDS. First,we modeled alkylator-induced t-AML/MDS by exposing wild type adult mice to N-ethyl-N-nitrosurea (ENU),resulting in several models of AML and MDS that have clinical and pathologic characteristics consistent with human t-AML/MDS including cytopenia,myelodysplasia,and shortened overall survival. These models were limited by their inability to transplant clinically aggressive disease. Second,we established three patient-derived xenograft models of human t-AML. These models led to rapidly fatal disease in recipient immunodeficient xenografted mice. LSC activity was identified in multiple HSPC subpopulations suggesting there is no canonical LSC immunophenotype in human t-AML. Overall,we report several new t-AML/MDS mouse models that could potentially be used to further define disease pathogenesis and test novel therapeutics. View Publication

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58{\%} Venus(+) HSPCs with 6-16{\%} human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD),caused by mutations in the gp91phox subunit of the NADPH oxidase,TI of a gp91phox transgene into AAVS1 resulted in ∼15{\%} gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs,4-11{\%} of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases. View Publication

This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells),metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material contain the differentiated luminal and basal cell types,whereas organoids derived from prostate cancer tissue mimic the histology of the tumor. We explain how to establish these cultures in the fully defined serum-free conditioned medium that is required to sustain organoid growth. Starting with the plating of digested tissue material,full-grown organoids can usually be obtained in ∼2 weeks. The culture protocol we describe here is currently the only one that allows the growth of both the luminal and basal prostatic epithelial lineages,as well as the growth of advanced prostate cancers. Organoids established using this protocol can be used to study many different aspects of prostate biology,including homeostasis,tumorigenesis and drug discovery. View Publication

Immunotherapy has increased overall survival of metastatic cancer patients,and cancer antigens are promising vaccine targets. To fulfill the promise,appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice,and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart,we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC,both important in human cancer development and progression,were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09,CASAC,Montanide ISA 51 VG,or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs),the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01,-1(*)07:02,-2(*)04:01,-2(*)05:02,and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-$\gamma$ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses,and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer. View Publication

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system,we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes,including TMPRSS2-ERG fusion,SPOP mutation,SPINK1 overexpression,and CHD1 loss. Whole-exome sequencing shows a low mutational burden,consistent with genomics studies,but with mutations in FOXA1 and PIK3R1,as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies. View Publication

Small-cell lung cancer (SCLC),an aggressive neuroendocrine tumor with early dissemination and dismal prognosis,accounts for 15-20{\%} of lung cancer cases and ∼200,000 deaths each year. Most cases are inoperable,and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs),which are prevalent in SCLC,present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice,and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged,and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms. View Publication

The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However,the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular,interferon-gamma$ (IFN-gamma$)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-gamma$ is commonly used to prime MSCs for immunomodulatory activity prior to transplantation,the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore,the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-gamma$ within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-gamma$ via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture,whereas IDO expression by IFN-gamma$-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore,sustained IDO expression induced by IFN-gamma$-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-gamma$-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether,microparticle delivery of IFN-gamma$ within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237. View Publication

Granulocyte colony-stimulating factor (G-CSF or CSF3) and its receptor CSF3R regulate granulopoiesis,neutrophil function,and hematopoietic stem cell mobilization. Recent studies have uncovered an oncogenic role of mutations in the CSF3R gene in many hematologic malignancies. To find additional CSF3R mutations that give rise to cell transformation,we performed a cellular transformation assay in which murine interleukin 3 (IL-3)-dependent Ba/F3 cells were transduced with WT CSF3R plasmid and screened for spontaneous growth in the absence of IL-3. Any outgrowth clones were sequenced to identify CSF3R mutations with transformation capacity. We identified several novel mutations and determined that they transform cells via four distinct mechanisms: 1) cysteine- and disulfide bond-mediated dimerization (S581C); 2) polar,noncharged amino acid substitution at the transmembrane helix dimer interface at residue Thr-640; 3) increased internalization by a Glu-524 substitution that mimics a low G-CSF dose; and 4) hydrophobic amino acid substitutions in the membrane-proximal residues Thr-612,Thr-615,and Thr-618. Furthermore,the change in signaling activation was related to an altered CSF3R localization. We also found that CSF3R-induced STAT3 and ERK activations require CSF3R internalization,whereas STAT5 activation occurred at the cell surface. Cumulatively,we have expanded the regions of the CSF3R extracellular and transmembrane domains in which missense mutations exhibit leukemogenic capacity and have further elucidated the mechanistic underpinnings that underlie altered CSF3R expression,dimerization,and signaling activation. View Publication

Cytoglobin (Cygb) is a member of the hemoglobin family and is thought to protect against cellular hypoxia and oxidative stress. These functions may be particularly important in inflammation-induced cancer,e.g.,in patients with ulcerative colitis (UC). In this study,we investigated the development of inflammation and tumors in a murine model of inflammation-induced colorectal cancer using a combined treatment of azoxymethane and dextran sulfate sodium. A bioinformatics analysis of genome-wide expression data revealed increased colonic inflammation at the molecular level accompanied by enhanced macroscopic tumor development in Cygb-deficient mice. Moreover,the expression of the UC-associated gene neurexophilin and PC-esterase domain family member 4 (Nxpe4) depended on the presence of Cygb in the inflamed colonic mucosa. Compared to wild type mice,RT-qPCR confirmed a 14-fold (p = 0.0003) decrease in Nxpe4 expression in the inflamed colonic mucosa from Cygb-deficient mice. An analysis of Cygb protein expression suggested that Cygb is expressed in fibroblast-like cells surrounding the colonic crypts. Histological examinations of early induced lesions suggested that the effect of Cygb is primarily at the level of tumor promotion. In conclusion,in this model,Cygb primarily seemed to inhibit the development of established microadenomas. View Publication

The intestinal microbiota has been identified as an environmental factor that markedly affects energy storage and body-fat accumulation in mammals,yet the underlying mechanisms remain unclear. Here we show that the microbiota regulates body composition through the circadian transcription factor NFIL3.Nfil3transcription oscillates diurnally in intestinal epithelial cells,and the amplitude of the circadian oscillation is controlled by the microbiota through group 3 innate lymphoid cells,STAT3 (signal transducer and activator of transcription 3),and the epithelial cell circadian clock. NFIL3 controls expression of a circadian lipid metabolic program and regulates lipid absorption and export in intestinal epithelial cells. These findings provide mechanistic insight into how the intestinal microbiota regulates body composition and establish NFIL3 as an essential molecular link among the microbiota,the circadian clock,and host metabolism. View Publication

BACKGROUND & AIMS Clostridium difficile induces intestinal inflammation by releasing toxins A and B. The antimicrobial compound cationic steroid antimicrobial 13 (CSA13) has been developed for treating gastrointestinal infections. The CSA13-Eudragit formulation can be given orally and releases CSA13 in the terminal ileum and colon. We investigated whether this form of CSA13 reduces C difficile infection (CDI) in mice. METHODS C57BL/6J mice were infected with C difficile on day 0,followed by subcutaneous administration of pure CSA13 or oral administration of CSA13-Eudragit (10 mg/kg/d for 10 days). Some mice were given intraperitoneal vancomycin (50 mg/kg daily) on days 0-4 and relapse was measured after antibiotic withdrawal. The mice were monitored until day 20; colon and fecal samples were collected on day 3 for analysis. Blood samples were collected for flow cytometry analyses. Fecal pellets were collected each day from mice injected with CSA13 and analyzed by high-performance liquid chromatography or 16S sequencing; feces were also homogenized in phosphate-buffered saline and fed to mice with CDI via gavage. RESULTS CDI of mice caused 60{\%} mortality,significant bodyweight loss,and colonic damage 3 days after infection; these events were prevented by subcutaneous injection of CSA13 or oral administration CSA13-Eudragit. There was reduced relapse of CDI after administration of CSA13 was stopped. Levels of CSA13 in feces from mice given CSA13-Eudragit were significantly higher than those of mice given subcutaneous CSA13. Subcutaneous and oral CSA13 each significantly increased the abundance of Peptostreptococcaceae bacteria and reduced the abundance of C difficile in fecal samples of mice. When feces from mice with CDI and given CSA13 were fed to mice with CDI that had not received CSA13,the recipient mice had significantly increased rates of survival. CSA13 reduced fecal levels of inflammatory metabolites (endocannabinoids) and increased fecal levels of 4 protective metabolites (ie,citrulline,3-aminoisobutyric acid,retinol,and ursodeoxycholic acid) in mice with CDI. Oral administration of these CSA13-dependent protective metabolites reduced the severity of CDI. CONCLUSIONS In studies of mice,we found the CSA13-Eudragit formulation to be effective in eradicating CDI by modulating the intestinal microbiota and metabolites. View Publication

Adequate availability of cellular building blocks,including lipids,is a prerequisite for cellular proliferation,but excess dietary lipids are linked to increased cancer risk. Despite these connections,specific regulatory relationships between membrane composition,intestinal stem cell (ISC) proliferation,and tumorigenesis are unclear. We reveal an unexpected link between membrane phospholipid remodeling and cholesterol biosynthesis and demonstrate that cholesterol itself acts as a mitogen for ISCs. Inhibition of the phospholipid-remodeling enzyme Lpcat3 increases membrane saturation and stimulates cholesterol biosynthesis,thereby driving ISC proliferation. Pharmacologic inhibition of cholesterol synthesis normalizes crypt hyperproliferation in Lpcat3-deficient organoids and mice. Conversely,increasing cellular cholesterol content stimulates crypt organoid growth,and providing excess dietary cholesterol or driving endogenous cholesterol synthesis through SREBP-2 expression promotes ISC proliferation in vivo. Finally,disruption of Lpcat3-dependent phospholipid and cholesterol homeostasis dramatically enhances tumor formation in Apcminmice. These findings identify a critical dietary-responsive phospholipid-cholesterol axis regulating ISC proliferation and tumorigenesis. View Publication