Serum Amyloid A3 Promoter-Driven Luciferase Activity Enables Visualization of Diabetic Kidney Disease

The early detection of diabetic nephropathy (DN) in mice is necessary for the development of drugs and functional foods. The purpose of this study was to identify genes that are significantly upregulated in the early stage of DN progression and develop a novel model to non-invasively monitor disease progression within living animals using in vivo imaging technology. Streptozotocin (STZ) treatment has been widely used as a DN model; however, it also exhibits direct cytotoxicity to the kidneys. As it is important to distinguish between DN-related and STZ-induced nephropathy, in this study, we compared renal responses induced by the diabetic milieu with two types of STZ models: multiple low-dose STZ injections with a high-fat diet and two moderate-dose STZ injections to induce DN. We found 221 genes whose expression was significantly altered during DN development in both models and identified serum amyloid A3 (Saa3) as a candidate gene. Next, we applied the Saa3 promoter-driven luciferase reporter (Saa3-promoter luc mice) to these two STZ models and performed in vivo bioluminescent imaging to monitor the progression of renal pathology. In this study, to further exclude the possibility that the in vivo bioluminescence signal is related to renal cytotoxicity by STZ treatment, we injected insulin into Saa3-promoter luc mice and showed that insulin treatment could downregulate renal inflammatory responses with a decreased signal intensity of in vivo bioluminescence imaging. These results strongly suggest that Saa3 promoter activity is a potent non-invasive indicator that can be used to monitor DN progression and explore therapeutic agents and functional foods.

A Novel In Vitro Method to Assess the Microbial Barrier Function of Tissue Adhesives Using Bioluminescence Imaging Technique

Background. Tissue glues can minimize treatment invasiveness, mitigate the risk of infection, and reduce surgery time; ergo, they have been developed and used in surgical procedures as wound closure devices beside sutures, staples, and metallic grafts. Regardless of their structure or function, tissue glues should show an acceptable microbial barrier function before being used in humans. This study proposes a novel in vitro method using Escherichia coli Lux and bioluminescence imaging technique to assess the microbial barrier function of tissue glues. Different volumes and concentrations of E. coli Lux were applied to precured or cured polyurethane-based tissue glue placed on agar plates. Plates were cultured for 1 h, 24 h, 48 h, and 72 h with bioluminescence signal measurement subsequently. Herein, protocol established a volume of 5 μL of a 1 : 100 dilution of E. coli Lux containing around 2 × 10 7 CFU/mL as optimal for testing polyurethane-based tissue glue. Measurement of OD600nm, determination of CFU/mL, and correlation with the bioluminescence measurement in p/s unit resulted in a good correlation between CFU/mL and p/s and demonstrated good reproducibility of our method. In addition, this in vitro method could show that the tested polyurethane-based tissue glue can provide a reasonable barrier against the microbial penetration and act as a bacterial barrier for up to 48 h with no penetration and up to 72 h with a low level of penetration through the material. Overall, we have established a novel, sensitive, and reproducible in vitro method using the bioluminescence imaging technique for testing the microbial barrier function of new tissue glues.

A chemerin peptide analog stimulates tumor growth in two xenograft mouse models of human colorectal carcinoma

Background: Chemerin plasma concentration has been reported to be positively correlated with the risk of colorectal cancer. However, the potential regulation of CRC tumorigenesis and progression has not yet been investigated in an experimental setting. This study addresses this hypothesis by investigating proliferation, colony formation and migration of CRC cell lines in vitro as well as in animal models. Methods: In vitro, microscopic assays to study proliferation as well as a scratch-wound assay for migration monitoring were applied using the human CRC cell lines HCT116, HT29 and SW620 under the influence of the chemerin analog CG34. The animal study investigated HCT116-luc and HT29-luc subcutaneous tumor size and bioluminescence during treatment with CG34 versus control, followed by ex-vivo analysis of vessel density and mitotic activity. Results: While proliferation of the three CRC cell lines in monolayers was not clearly stimulated by CG34, the chemerin analog promoted colony formation in three-dimensional aggregates. An effect on cell migration was not observed. In the treatment study, CG34 significantly stimulated both growth and bioluminescence signal of HCT116-luc and HT29-luc xenografts. Conclusions: The results of this study represent the first indication of a tumor growth-stimulating effect of chemerin signaling in CRC.

Using the Microwell-mesh to culture microtissues in vitro and as a carrier to implant microtissues in vivo into mice

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of “pieces” of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ−/− (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 μm achieving superior microtissue vascularisation relative to mesh with 36×36 μm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.

A survivin-driven tumour-activatable minicircle system for prostate cancer theranostics

Gene vectors driven by tumour-specific promoters to express reporter genes and therapeutic genes are an emerging approach for improved cancer diagnosis and treatment. Minicircles (MCs) are shortened plasmids stripped of prokaryotic sequences and have potency and safety characteristics that are beneficial for clinical translation. We previously developed survivin-driven, tumour-activatable MCs for cancer detection via a secreted blood reporter assay. Here we present a novel theranostic system for prostate cancer featuring a pair of survivin-driven MCs, combining selective detection of aggressive tumours via a urinary reporter test and subsequent tumour treatment with gene-directed enzyme prodrug therapy.MethodsWe engineered both diagnostic and therapeutic survivin-driven MCs expressing Gaussia luciferase, a secreted reporter that is detectable in the urine, and cytosine deaminase:uracil phosphoribosyltransferase fusion, respectively. Diagnostic MCs were evaluated in mice carrying orthotopic prostate tumours with varying survivin levels, measuring reporter activity in serial urine samples. Therapeutic MCs were evaluated in mice receiving prodrug using bioluminescence imaging to assess cancer cell viability over time.ResultsDiagnostic MCs revealed mice with aggressive prostate tumours exhibited significantly higher urine reporter activity than mice with non-aggressive tumours and tumour-free mice. Combined with 5-fluorocytosine prodrug treatment, therapeutic MCs resulted in reduced bioluminescence signal in mice with aggressive prostate tumours compared to control mice.ConclusionSequential use of these MCs may be used to first identify patients carrying aggressive prostate cancer by a urinary reporter test, followed by stringent treatment in stratified individuals identified to have high-risk lesions. This work serves to highlight tumour-activatable MCs as a viable platform for development of gene-based tumour-activatable theranostics.

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

Bioluminescence-based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has, so far, not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterialluxCDABEandfrpgenes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.

Imaging of red-shifted photons from bioluminescent tumours using fluorescence by unbound excitation from luminescence

Early detection of tumours is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. Bioluminescence imaging has been widely used in the field of oncology and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, photons that are emitted by the majority of commonly used luciferases are usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal and calling for optimised optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection in vivo. Here, we applied FUEL to tumour detection in two different subcutaneous tumour models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with the plasmid Luc2. Tumour size and bioluminescence were measured over time and tumour vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs)in the tumour site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumour detection.

Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice

ABSTRACTReporter viruses provide a powerful tool to study infection, yet incorporating a nonessential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus withNanoLuc luciferaseinserted into the 5′ end of the PA gene segment grew to titers 10-fold less than those of the wild type in MDCK cells and in DBA/2 mice and was less virulent. For 12 rounds, we propagated DBA/2 lung samples with the highest bioluminescence-to-titer ratios. Every three rounds, we comparedin vivoreplication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino acid mutations andPB2noncoding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate that directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-likein vivofitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCEInfluenza viruses contribute to 290,000 to 650,000 deaths globally each year. Infection is studied in mice to learn how the virus causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene that causes infected cells to light up, scientists could see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza virus infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.

Comparative Analysis Between the In Vivo Biodistribution and Therapeutic Efficacy of Adipose-Derived Mesenchymal Stromal Cells Administered Intraperitoneally in Experimental Colitis

Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. It is described that the immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless the route of administration used, the inflammation conditions and the MHC context. These observations arise the question as to whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the `responder´ mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial to achieve an optimal modulation of inflammation.

5-Azacytidine Prevents Relapse from Discrete Bone Marrow Sites in Leukemia Xenografts Treated with CD22-Targeting Immunotoxin Moxetumomab Pasudotox

Background: Moxetumomab pasudotox (Moxe) is a recombinant immunotoxin that targets CD22. Moxe showed high clinical activity in a phase II trial against hairy cell leukemia (Kreitman et al, 2012) and a phase III trial has recently completed enrollment. Moxe also demonstrated objective responses in 30% of children with multiply relapsed acute lymphoblastic leukemia (ALL) (Wayne et al AACR 2014). In a xenograft model with the ALL cell line KOPN-8, Moxe as monotherapy clears the murine bone marrow (BM) from leukemia blasts (Mueller et al, CCR 2016). However, mice eventually relapse and die of progressive disease. Goal: To characterize the ALL cells responsible for relapse and to devise a relapse-preventing treatment. Methods/Results: The ALL cell lines KOPN-8 and REH were transduced with luciferase. The systemic bioluminescence signal in KOPN-8 bearing mice was abrogated after three i.v. bolus-doses of Moxe. Relapse was subsequently detected at various, apparently randomly distributed BM sites which possibly were the origin of systemic relapse. The systemic relapse remained Moxe-sensitive for several treatment cycles, but the bioluminescence signal at the discrete BM sites persisted. After several treatment cycles a Moxe-resistant systemic relapse emerged and mice died of progressive disease as determined by bioluminescence and flow cytometry. The Moxe-resistant cells showed a loss of CD10, and a 2.5-fold decrease in surface CD19 and CD22. CD22 mRNA levels and total CD22 protein by Western Blot were reduced up to 7-fold, indicating a profound reduction of intracellular CD22 in resistant cells. RNA deep sequencing revealed a global transcriptional change including down-regulation of B-cell specific genes suggesting a less mature state of KOPN-8 in the Moxe resistant blasts. We had previsouly demonstrated an epigenetic mechanism of Moxe resistance in vitro (Hu et al, Leuk Res 2013). Because most of the genetically homogeneous KOPN-8 cells growing in murine BM were Moxe sensitive but cells at discrete sites persisted, we hypothesized that locally induced epigenetic changes might partially explain the resistance. We therefore combined Moxe with the DNA-methyltransferase inhibitor 5-Azacytidine (5-AZA). KOPN-8 bearing mice treated with Moxe or 5-AZA alone survived longer than untreated mice, but eventually died from leukemia. However, mice that received the combination of 5-AZA and Moxe were disease free at 4 months after treatment by flow cytometry and histopathology. The combination of 5-AZA and Moxe also improved responses over each agent alone in a second ALL xenograft model with the ALL cell line REH. Conclusion: The relapse of KOPN-8 after treatment with Moxe, possibly from discrete BM sites is consistent with BM-niche induced resistance. This niche-induced resistance is further supported by locally persisting cells after repeated treatment cycles, while blasts that repopulate other BM sites remain rIT-sensitive. That relapse at discrete BM-sites is prevented by 5-AZA suggests that epigenetic modifications contribute to induction of localized resistance in vivo. rIT-resistant KOPN-8 relapse shows a profound reduction in total CD22 which may contribute to resistance to CD22-directed rIT. The marked increase of Moxeefficacy in vivo by the addition of 5-AZA makes this combination a promising approach for further clinical testing in children and adults with ALL. Disclosures Wayne: Kite Pharma: Honoraria, Other: Travel support, Research Funding; Spectrum Pharmaceuticals: Honoraria, Other: Travel Support, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support; Medimmune: Honoraria, Other: Travel Support, Research Funding; NIH: Patents & Royalties. Pastan:NIH: Patents & Royalties: Co-Inventor on NIH Patent.