Whole-body Bioluminescence Imaging Research Articles

Cerclage prevents ascending intrauterine infection in pregnant mice

The treatment for cervical insufficiency is cerclage surgery. Although cerclage is a common therapy for prevention of preterm birth, there is no consensus about its mechanism of efficacy. Previous investigators have hypothesized that cerclage prevents preterm birth by improving the cervical barrier to ascending infection. However, this hypothesis is difficult to study in human pregnancy. In a mouse model of ascending infection, we hypothesized that a cerclage improves the cervical barrier leading to decreased ascending intrauterine infection and inflammation. An abdominal cerclage was studied because a vaginal cerclage is not feasible in mice. To perform an abdominal cerclage, laparotomy was performed on timed, pregnant C57BL/6 mice on gestational day 10 (E10). A 6-0 silk suture was placed around the cervix just below the junction of the 2 uterine horns. Sham controls received the same surgery, but no cerclage was placed. To track ascending infection nonpathogenic E coli K12 was genetically modified to express bioluminescence. On E15, bioluminescent E coli K12 (20 μL of 1×109 bacteria) was inoculated into the vagina. Whole-body bioluminescence imaging was performed 0.5 hours and 24 hours after inoculation. To assess intrauterine inflammation, pathogenic E coli K1 was used. On E15, bacterial inoculums of E coli K1 (20 μL of 1×104 bacteria) were vaginally administered. Samples of uterus, placenta, and fetal membranes were collected 24 hours after inoculation. Gene expression of inflammation-related proteins was compared between 3 groups: (1) sham control surgery+ inoculation of phosphate-buffered saline (PBS), (2) sham control surgery+ inoculation of E coli K1, and (3) cerclage surgery+ inoculation of E coli K1. Abdominal cerclage was well tolerated. No cases of preterm birth were seen following abdominal cerclage. Whole-body bioluminescent imaging performed 0.5 hours post inoculation showed a strong luminescence signal in the vaginal region of mice in both control and experimental groups indicating successful bacteria inoculation. Twenty-four hours after inoculation, bioluminescent signal was seen ascending into the uterine horns in all control mice. However, in mice with abdominal cerclages, no bioluminescent signal was seen after 24 hours. When the reproductive tissues were imaged separately in control mice, strong bioluminescence signal was detected in the placenta, fetal membranes, and uterus. Gene expression studies showed that cerclage significantly decreased the expression of inflammatory proteins induced by E coli K1 in the uterus, placenta, and fetal membranes. In this mouse model of ascending intrauterine infection, abdominal cerclage prevented ascending infection of E coli. In addition, abdominal cerclage prevented expression of inflammatory cytokines in the uterus, placenta, and membranes of mice. The study provides evidence for a potential mechanism of cerclage success in human pregnancy.

Phonozen-mediated photodynamic therapy comparing two wavelengths in a mouse model of peritoneal carcinomatosis.

This study assessed the therapeutic efficacy of intraperitoneal photodynamic therapy (PDT) using photosensitizer activation at two different wavelengths, 405 and 664nm, in a mouse model of peritoneal carcinomatosis. The dark and light cytotoxicity of chlorin e6-polyvinylpyrrolidone (Phonozen) were measured in vitro under 402 ± 14 and 670 ± 18nm LED activation in bioluminescent human gastric cancer cells, MKN45-luc. Cell viability was measured at 6h after irradiation using the PrestoBlue assay. Corresponding in vivo studies were performed in athymic nude mice by intraperitoneal injection of 1 × 106 MKN45-luc cells. PDT was performed 10 d after tumor induction and comprised intraperitoneal injection of Phonozen followed by light irradiation at 3h, delivered by a diffusing-tip optical fiber placed in the peritoneal cavity and coupled to a 405 or 664nm diode laser to deliver a total energy of 50J (20 mice per cohort). Whole-body bioluminescence imaging was used to track the tumor burden after PDT out to 130days, and 5 mice in each cohort were sacrificed at 4h post treatment to measure the acute tumor necrosis. Photosensitizer dose-dependent photocytotoxicity was higher in vitro at 405 than 664nm. In vivo, PDT reduced the tumor growth rate at both wavelengths, with no statistically significant difference. There was substantial necrosis, and median survival was significantly prolonged at both wavelengths compared with controls (46 and 46 vs. 34 days). Phonozen-mediated PDT results in significant cytotoxicity in vitro as well as tumor necrosis and prolonged survival in vivo following intraperitoneal light irradiation. Blue light was more photocytotoxic than red in vitro and had marginally higher efficacy in vivo.

Whole-body bioluminescence imaging of T-cell response in PDAC models.

The location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo. Here, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically 'hot') or low T-cell infiltration (immunologically 'cold'). Differences in total T-cell bioluminescence could be seen between the 'hot' and 'cold' tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment. In the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Label-free metabolic imaging for sensitive and robust monitoring of anti-CD47 immunotherapy response in triple-negative breast cancer

BackgroundImmunotherapy is revolutionizing cancer treatment from conventional radiotherapies and chemotherapies to immune checkpoint inhibitors which use patients’ immune system to recognize and attack cancer cells. Despite the huge clinical success...

Abstract 1075: Therapeutic targeting of CBFA2T3-GLIS2 infant AML with ELU001 - folate receptor alpha-directed C’Dot-drug-conjugate

Abstract Acute myeloid leukemia (AML) in infants is a unique subtype of AML that is driven by oncogenic fusions, resulting in a highly proliferative disease. The CBFA2T3-GLIS2 oncogenic fusion that is seen exclusively in infants and young children <3 years is the most refractory AML with near uniform mortality with conventional therapies. As part of our Target Pediatric AML (TpAML) discovery effort to define novel therapeutic targets, we discovered folate receptor alpha (FRα, FOLR1) transcript to be expressed in CBF-GLIS positive AML and verified cell surface expression of FOLR1 on leukemic cells. We present pre-clinical efficacy of a novel FOLR1 directed therapy (ELU001) in CBF-GLIS AML.ELU001 is a novel ~6 nm-diameter FOLR1-targeted C’Dot-Drug-Conjugate (CDC) incorporating ~21 exatecan molecules as a payload that is designed to treat cancers with improved safety and efficacy as compared to antibody-drug conjugates. A Phase 1 clinical trial of ELU001 in adult patients with tumors that overexpress FOLR1 is currently underway (NCT05001282). In this study, we determined the potential utility of ELU001 for CBFA2T3-GLIS2 AML by evaluating its preclinical efficacy in AML models. We confirmed the binding of ELU001 to AML cells expressing FOLR1 by flow cytometry using MV4;11 AML cell line transduced with an FOLR1 expression construct (MV4;11 FOLR1+). We show that MV4;11 FOLR1+ cells readily bind to ELU001 but not MV4;11 parental cells. Consistent with target-dependent binding, in vitro cytotoxicity studies demonstrated that MV4;11 FOLR1+ cells were highly susceptible to ELU001 (IC50=30 pM) while MV4;11 parental cells were only minimally affected by treatment. To evaluate in vivo efficacy, we transplanted MV4;11 FOLR1+ cells transduced with Luciferase-expression construct into immune deficient NSG mice at 1M cells per mouse and treated the mice the following week with different doses of ELU001 (vehicle, 0.3mg/kg Q3Dx6, 0.5 mg/kg Q3Dx3, and 0.65mg/kg Q3Dx3). Leukemia burden was assessed by whole-body bioluminescence (IVIS) imaging, performed weekly. IVIS imaging showed that ELU001 effectively eradicated the leukemia in vivoat all three doses tested resulting in enhanced survival while mice that received the vehicle treatment exhibited disease progression and eventually succumb to the disease. We confirmed that the leukemia was absent in ELU001-treated mice at the study endpoint of 120 days post treatment by flow cytometric analysis of the bone marrow, spleen and liver tissues harvested at necropsy. ELU001 was well tolerated with an apparent maximum tolerance dose of 1.50 mg/kg/mouse (0.5 mg/kg exatecan, Q3DX3). In summary, we demonstrated that ELU001 is highly effective at eliminating FOLR1 positive AML cells in vitro and in vivo, providing the preclinical data to support further assessment of ELU001 in clinical trials for pediatric patients with CBFA2T3-GLIS2 fusions overexpressing FOLR1. Citation Format: Quy Le, Feng Chen, Thao Tang, Cynthia Nourigat McKay, Fei Wu, Melik Turker, Thomas Gardinier, Vaibhav Patel, Aranapakam Venkatesan, Tin Khor, Kai Ma, Gregory P. Adams, Soheil Meshinchi. Therapeutic targeting of CBFA2T3-GLIS2 infant AML with ELU001 - folate receptor alpha-directed C’Dot-drug-conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1075.

Reducing regorafenib toxicity by combining with dual JAK-HDAC inhibitor in colorectal cancer.

e15597 Background: In the US, colorectal cancer (CRC) is the third most common cancer. Patients receiving regorafenib, a multiple-kinase inhibitor, recommended to manage metastatic CRCs (mCRCs), has a modest improvement in median overall survival but it is associated with several toxicities. Our present study addresses regorafenib-induced toxicity concerns by combining regorafenib with a novel dual JAK-HDAC inhibitor (JAK-HDACi). The rationale for the dual inhibitor drug selection is due to the facts that the JAK/STAT/SOCS pathway is modulated in CRCs, and concurrent inhibition of JAK sensitizes solid tumors to HDACi. This study focused on evaluating the efficacy and reducing regorafenib-induced toxicity with this novel therapeutic combination in CRC preclinical models. Methods: We evaluated the toxicity of the JAK-HDACi, regorafenib, and their combination in normal colonic cells (CRL-1807) and their efficacy in CRC cell lines (HCT116, RKO, HT29, and SW480) exhibiting various statuses of p53, KRAS, BRAF, EGFR, and microsatellite instability, by conducting colony formation, cell proliferation, and cell cycle arrest assays. Kinome profiling and whole transcriptomic analysis were performed. Their efficacy was assessed in vivo in a CRC patient-derived xenograft (PDX) model, and experimental metastasis was evaluated in NSG mice using luciferase-tagged HT29 cells. Non-invasive, whole-body bioluminescence imaging was performed. Tumor tissues were harvested and stored at −80°C or prepared formalin-fixed paraffin-embedded blocks for Hematoxylin and Eosin (H&E) and immunostaining. Serum analysis was performed to evaluate liver and kidney functions to assess the toxicity. Results: At 500 nM concentrations, there was no pronounced death of CRL-1807 cells, but reduced number of colonies in CRC cells. Drug treatments decreased phosphorylation of STAT3 and ERK1/2 and cell viability, wherein the reduction was robust in the combination. The combination reduced activity of various kinases, as evident through kinome profiling. In SW480 cells, the combination caused G0-G1 cell arrest and decreased the S phase. RNA-seq results revealed modulation of key pathways: apoptosis, ECM-receptor interaction, and focal adhesion. The PDX model showed that the combination treatment reduced tumor growth, as evidenced in H&E staining with higher necrosis and reduced Ki67 staining. Experimental metastasis, bioluminescence imaging, and histological examination showed pronounced reduction in metastasis in mice treated with the combination. Serum chemistry profiles showed that the treatments did not cause systemic toxicity to mice used in either model. Conclusions: The combination therapy with the JAK-HDACi and regorafenib was more effective than the single agents with no evident toxicity. These findings lend credence to a clinical trial to assess this combination for treatment of patients with advanced CRC.

Adriamycin/Nucleophosmin Binding Protein-Conjugated Nanoparticle (ADR-PMs-NPMBP) Enhances Anti-Leukemia Activities of Adriamycin in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is an aggressive malignancy. Adults with ALL have more than 50% relapse rates and 20-40% overall survival. We previously validated that overexpression of NPM may be involved in the multidrug resistance development and might be important indicator for prognosis evaluation in ALL. In this study, we engineered and synthesized recombinant NPM binding protein (NPMBP). NPMBP and adriamycin (ADR) were bound to polymeric nanoparticle-based drug delivery system and named ADR-PMs-NPMBP (China Patent Application 202010302395.6, 202010739575.0). In this study, we aimed to evaluate thein vitroandin vivoanti-leukemia activity of ADR-PMs-NPMBP in resistant ALL cells. The underlying mechanisms were further addressed. In vitrostudy data demonstrated that ADR-PMs-NPMBP significantly enhanced chemosensitivity of ALL cells to ADR. The IC50 values of ADR-PMs-NPMBP in 4 ALL cell lines and primary ALL cells from 6 patients were between 1.6- and 7.0-fold lower than those of ADR. Of note, all the resistant ALL cell lines and the primary ALL cells from the relapsed patients, although variable in degree, were sensitive to ADR-PMs-NPMBP. The ratio of apoptosis cells in resistant ALL cells was more than 2-fold as compared ADR-PMs-NPMBP with single ADR regimen. RNA-seq analysis showed that there were 5599 and 233 differentially expressed genes in ADR-PMs-NPMBP versus control and ADR-PMs-NPMBP versus ADR comparison groups, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that 21 pathways play significant roles in ADR-PMs-NPMBP-mediated anti-leukemia activities in ALL. Of note, p53 protein with gene symbol TP53 was interestingly identified to overlap in the 5 pathways as following: Epstein-Barr virus infection, cell cycle, apoptosis, human T-cell leukemia virus 1 infection and p53 signaling pathway. Further validation experiments showed that ADR-PMs-NPMBP-induced effects on cell viability were due to the multiple roles of the activated p53 in apoptosis induction and cell cycle arrest. In vivostudy showed that ADR-PMs-NPMBP regimen was well tolerated by both resistant ALL xenograft BALB/C-nude model and ALL cell transplanted NOD-Prkdcem26Il2rgem26Nju (NCG) model. No systemic toxicity occurrence was observed after the recipients were intraperitoneally administered with ADR-PMs-NPMBP at 25 mg/kg/day for 3 days. The body weights of the animals increased with the time in both tumor model systems. Of note, whole-body bioluminescence imaging showed that less tumor cell burden was observed in ADR-PMs-NPMBP-treated recipients as compared to the animals administered with ADR. The tumor inhibition rate achieved as high as 79.2% in ADR-PMs-NPMBP group when finished the following-up. Moreover, studies on NCG model showed that ADR-PMs-NPMBP conditioning sensitized the antileukemia response of the resistant ALL xenograft model to ADR. Wright-Giemsa staining results demonstrated that more mature myeloid cells and lymphocytes were presented in the bone marrow samples from ADR-PMs-NPMBP-conditioned mice. In contrast, the increased immature blast cells were observed in the bone marrow from vehicle control mice and ADR-treated mice. Notably, the regimen of ADR-PMs-NPMBP diminished or blocked simultaneous metastases to multiple organs including liver and spleen, and significantly prolonged mouse survival. In conclusion, our study provides the first evidence that ADR-PMs-NPMBP nanoparticle may exert apoptotic inductionviathe activation of p53 pathway and markedly improve the antileukemia effect of ADR in resistant ALL cellsin vitroandin vivo. Our data indicate that the novel drug delivery system may be valuable to develop as a new strategy for the treatment of ALL, particularly for the resistant ALL. Disclosures No relevant conflicts of interest to declare.

Non-invasive somatotransgenic bioimaging in living animals

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.

In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei.

Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei brucei parasitemia in the blood. Testing the efficacy of new compounds in these models is cumbersome because it may take several months after treatment before surviving parasites become detectable in the blood. To expedite compound screening, we have used a Trypanosoma brucei brucei GVR35 strain expressing red-shifted firefly luciferase to monitor parasite distribution in infected mice through noninvasive whole-body bioluminescence imaging. This protocol describes the infection and in vivo bioluminescence imaging of mice to assess compound efficacy against T. brucei during the two characteristic stages of disease, the hemolymphatic phase (stage 1) and the encephalitic or central nervous system phase (stage 2).

Sustained activity of novel THIOMAB antibody-antibiotic conjugate against Staphylococcus aureus in a mouse model: Longitudinal pharmacodynamic assessment by bioluminescence imaging.

Staphylococcus aureus (S. aureus) infections are a leading cause of death by an infectious agent. Survival within host phagocytic cells is one mechanism by which S. aureus evades antibiotic treatment. A novel THIOMAB™ antibody-antibiotic conjugate (TAC) strategy was developed to kill S. aureus intracellularly and mitigate the spread of infection. In this report, we used a longitudinal whole-body bioluminescence imaging method to study the antibacterial dynamics of TAC alone or in combination with vancomycin in a mouse infection model. Injections of stably luminescent S. aureus bacteria into mice resulted in exponential increases in whole body bioluminescence with a reduction in body weight and survival rate. Vancomycin, a standard-of-care antibiotic, suppressed bacterial growth in mice. However, bacterial growth rebounded in these animals once treatment was discontinued. In contrast, single dose of TAC showed rapid reduction of bioluminescence intensity, which persisted for up to 19 days. The combination of TAC and vancomycin achieved a more sustained and significantly greater reduction of bioluminescence compared with vancomycin alone. In summary, the present study showed an imaging method to longitudinally assess antibacterial drug dynamics in mice and demonstrated that TAC monotherapy or in combination with vancomycin had superior and sustained activity compared to vancomycin alone.

Updating the modified Thompson test by using whole-body bioluminescence imaging to replace traditional efficacy testing in experimental models of murine malaria

BackgroundRodent malaria models are extensively used to predict treatment outcomes in human infections. There is a constant need to improve and refine these models by innovating ways to apply new scientific findings and cutting edge technologies. In addition, and in accordance with the three R’s of animal use in research, in vivo studies should be constantly refined to avoid unnecessary pain and distress to the experimental animals by using preemptive euthanasia as soon as the main scientific study objective has been accomplished.MethodsThe new methodology described in this manuscript uses the whole-body bioluminescence signal emitted by transgenic, luciferase-expressing Plasmodium berghei parasites to assess the parasite load predicted parasitaemia (PLPP) in drug and control treated female ICR-CD1 mice infected with 1 × 105 luciferase-expressing P. berghei (ANKA strain) infected erythrocytes. This methodology can replace other time-consuming and expensive methods that are routinely used to measure parasitaemia in infected animals, such as Giemsa-stained thin blood smears and flow cytometry.ResultsThere is a good correlation between whole-body bioluminescence signal and parasitaemia measured using Giemsa-stained thin blood smears and flow cytometry respectively in donor and study mice in the modified Thompson test. The algebraic formulas which represent these correlations can be successfully used to assess PLPP in donor and study mice. In addition, the new methodology can pinpoint sick animals 2–8 days before they would have been otherwise diagnosed based on behavioural or any other signs of malaria disease.ConclusionsThe new method for predicting parasitaemia in the modified Thompson test is simple, precise, objective, and minimizes false positive results that can lead to the premature removal of animals from study. Furthermore, from the animal welfare perspective of replace, reduce, and refine, this new method facilitates early removal of sick animals from study as soon as the study objective has been achieved, in many cases well before the clinical signs of disease are present.

Production of lentiviral vectors using novel, enzymatically produced, linear DNA

The manufacture of large quantities of high-quality DNA is a major bottleneck in the production of viral vectors for gene therapy. Touchlight Genetics has developed a proprietary abiological technology that addresses the major issues in commercial DNA supply. The technology uses ‘rolling-circle’ amplification to produce large quantities of concatameric DNA that is then processed to create closed linear double-stranded DNA by enzymatic digestion. This novel form of DNA, Doggybone™ DNA (dbDNA™), is structurally distinct from plasmid DNA. Here we compare lentiviral vectors production from dbDNA™ and plasmid DNA. Lentiviral vectors were administered to neonatal mice via intracerebroventricular injection. Luciferase expression was quantified in conscious mice continually by whole-body bioluminescent imaging. We observed long-term luciferase expression using dbDNA™-derived vectors, which was comparable to plasmid-derived lentivirus vectors. Here we have demonstrated that functional lentiviral vectors can be produced using the novel dbDNA™ configuration for delivery in vitro and in vivo. Importantly, this could enable lentiviral vector packaging of complex DNA sequences that have previously been incompatible with bacterial propagation systems, as dbDNA™ technology could circumvent such restrictions through its phi29-based rolling-circle amplification.

Ascending Vaginal Infection Using Bioluminescent Bacteria Evokes Intrauterine Inflammation, Preterm Birth, and Neonatal Brain Injury in Pregnant Mice

Preterm birth is a serious global health problem and the leading cause of infant death before 5 years of age. At least 40% of cases are associated with infection. The most common way for pathogens to access the uterine cavity is by ascending from the vagina. Bioluminescent pathogens have revolutionized the understanding of infectious diseases. We hypothesized that bioluminescent Escherichia coli can be used to track and monitor ascending vaginal infections. Two bioluminescent strains were studied: E. coli K12 MG1655-lux, a nonpathogenic laboratory strain, and E. coli K1 A192PP-lux2, a pathogenic strain capable of causing neonatal meningitis and sepsis in neonatal rats. On embryonic day 16, mice received intravaginal E. coli K12, E. coli K1, or phosphate-buffered saline followed by whole-body bioluminescent imaging. In both cases, intravaginal delivery of E. coli K12 or E. coli K1 led to bacterial ascension into the uterine cavity, but only E. coli K1 induced preterm parturition. Intravaginal administration of E. coli K1 significantly reduced the proportion of pups born alive compared with E. coli K12 and phosphate-buffered saline controls. However, in both groups of viable pups born after bacterial inoculation, there was evidence of comparable brain inflammation by postnatal day 6. This study ascribes specific mechanisms by which exposure to intrauterine bacteria leads to premature delivery and neurologic inflammation in neonates.

Dysregulated HAI-2 Plays an Important Role in Renal Cell Carcinoma Bone Metastasis through Ligand-Dependent MET Phosphorylation.

MET, a c-met proto-oncogene product and hepatocyte growth factor (HGF) receptor, is known to play an important role in cancer progression, including bone metastasis. In a previous study, we reported increased expression of MET and matriptase, a novel activator of HGF, in bone metastasis. In this study, we employed a mouse model of renal cell carcinoma (RCC) bone metastasis to clarify the significance of the HGF/MET signaling axis and the regulator of HGF activator inhibitor type-2 (HAI-2). Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of mice to prepare the mouse model of bone metastasis. The formation of bone metastasis was confirmed by whole-body bioluminescent imaging, and specimens were extracted. Expression of HGF/MET-related molecules was analyzed. Based on the results, we produced HAI-2 stable knockdown 786-O cells, and analyzed invasiveness and motility. Expression of HGF and matriptase was increased in bone metastasis compared with the control, while that of HAI-2 was decreased. Furthermore, we confirmed increased phosphorylation of MET in bone metastasis. The expression of matriptase was upregulated, and both invasiveness and motility were increased significantly by knockdown of HAI-2. The significance of ligand-dependent MET activation in RCC bone metastasis is considered, and HAI-2 may be an important regulator in this system.

Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells.

Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major. Significant infiltrates of neutrophils and monocytes occurred at 1–4 d and 2–4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-γ in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.—Goundry, A., Romano, A., Lima, A. P. C. A., Mottram, J. C., Myburgh, E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells.

Gambogic Acid Efficiently Kills Stem-Like Colorectal Cancer Cells by Upregulating ZFP36 Expression

Background/Aims: Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported to be a potential novel antitumor drug. Whether GA inhibits putative cancer stem cells (CSCs), which are considered to be the major cause of cancer treatment failure, remains largely unknown. This study investigated whether GA inhibits the CSCs of colorectal cancer (CRC) and its possible mechanisms. Methods: We performed CCK8 and tumor sphere formation assays, percentage analysis of both side population and CD133⁺CD44⁺ cells, and the detection of stem cells markers, in order to assess the role of GA in inhibiting the stem celllike features of CRC. An mRNA microarray was performed to identify the downstream gene affected by GA and rescue assays were performed to further clarify whether the downstream gene is involved in the GA induced decrease of the stem cell-like CRC population. CRC cells were engineered with a CSC detector vector encoding GFP and luciferase (Luc) under the control of the Nanog promoter, which were utilized to investigate the effect of GA on putative CSC in human tumor xenograft-bearing mice using in vivo bioluminescence imaging. Results: Our results showed that GA significantly reduced tumor sphere formation and the percentages of side population and CD133⁺CD44⁺ cells, while also decreasing the expression of stemness and EMT-associated markers in CRC cells in vitro. GA killed stem-like CRC cells by upregulating the expression of ZFP36, which is dependent on the inactivation of the EGFR/ ERK signaling pathway. GFP+ cells harboring the P_Nanog-GFP-T2A-Luc transgene exhibited CSC characteristics. The in vivo results showed that GA significantly inhibited tumor growth in nude mice, accompanied by a remarkable reduction in the putative CSC number, based on whole-body bioluminescence imaging. Conclusion: These findings suggest that GA significantly inhibits putative CSCs of CRC both in vitro and in vivo by inhibiting the activation of the EGFR/ ERK/ZFP36 signaling pathway and may be an effective drug candidate for anticancer therapies.

In Vivo Bioluminescence Imaging of Intratumoral Bacteria.

This chapter describes the use of whole-body bioluminescent imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in therapy of cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and tumor sites concurrently. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor-bearing mice. This powerful, and inexpensive, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research. This protocol outlines the procedure for studying lux-tagged Escherichia coli and Bifidobacterium breve in mice, demonstrating the spatial and temporal readout from 2D and 3D BLI achievable with whole-body in vivo luminescence imaging.

Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition.

Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment.