Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

A method for non-invasive visualization of genetically labelled cells in animal disease models with micron-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the “optical window” above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune, previously the brightest monomeric FP when excited beyond 600 nm. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts and stem cells into myocytes in living mice with high anatomical detail

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2)) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH(2) supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

Background: Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the colorcoupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Principal Findings: Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. Significance: We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays

Temporal Variations of Skin Pigmentation in C57Bl/6 Mice Affect Optical Bioluminescence Quantitation

ABSTRACT PURPOSE: Depilation-induced skin pigmentation in C57Bl/6 mice is a known occurrence, and presents a unique problem for quantitative optical imaging of small animals, especially for bioluminescence. The work reported here quantitatively investigated the optical attenuation of bioluminescent light due to melanin pigmentation in the skin of transgenic C57B1/6 mice, modified such that luciferase expression is under the transcription control of a physiologically and pharmacologically inducible gene. PROCEDURE: Both in vivo and ex vivo experiments were performed to track bioluminescence signal attenuation through different stages of the mouse hair growth cycle. Simultaneous reflectance measurements were collected in vivo to estimate melanin levels. RESULTS: Biological variability of skin pigmentation was found to dramatically affect collected bioluminescent signal emerging through the skin of the mice. When compared to signal through skin with no pigmentation, the signal through highly-pigmented skin was attenuated an average of 90%. Correlation of reflectance signals to bioluminescence signal loss forms the basis of the proposed correction method. We observed, however, that variability in tissue composition, which results in inconsistent reflectance spectra, limits the accuracy of the correction method but can be improved by incorporating more complex analysis. CONCLUSION: Skin pigmentation is a significant variable in bioluminescent imaging, and should be considered in experimental design and implementation for longitudinal studies, and especially when sensitivity to small signal changes, or differences among animals, is required

In vivo bioluminescence imaging of locally disseminated colon carcinoma in rats

Animal tumour models using orthotopic tumours for the evaluation of cancer therapies are of greater clinical relevance than subcutaneous models, but they also pose greater difficulties for measuring tumour size and quantifying response to treatment. In this study, we used noninvasive bioluminescence imaging to monitor the intraperitoneal growth of luciferase-transfected CC531 colorectal cells in adult WAG/RIJ rats. The bioluminescence signal correlated well with post-mortem assessment of tumour load by visual inspection of the peritoneal cavity at specific follow-up times. Using bioluminescence imaging, we were able to monitor peritoneal tumour growth sequentially in time and to calculate a tumour growth rate for each animal; this is not possible with invasive methods of evaluating tumour load. Bioluminescence imaging of rats treated with a single dose of cisplatin (4 mg x kg(-1), i.p.) demonstrated a significant delay in peritoneal tumour growth relative to saline controls (mean 45.0+/-s.d. 13.0 vs 28.2+/-10.3 days; P=0.04). Similar protocols evaluated by visual scoring of tumour load at 40 days after inoculation supported these findings, although no quantitative assessment of treatment-induced growth delay could be made by this method. This study shows that in vivo imaging of luciferase-transfected tumour cells is a useful tool to investigate the dynamics of disseminated tumour growth and efficacy of anticancer treatment in orthotopic models of peritoneal cancer in rats. It offers an attractive alternative to invasive methods, and requires fewer animals for measuring tumour response to therapy

Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice

INTRODUCTION: Our goal was to generate xenograft mouse models of human breast cancer based on luciferase-expressing MDA-MB-231 tumor cells that would provide rapid mammary tumor growth; produce metastasis to clinically relevant tissues such as lymph nodes, lung, and bone; and permit sensitive in vivo detection of both primary and secondary tumor sites by bioluminescent imaging. METHOD: Two clonal cell sublines of human MDA-MB-231 cells that stably expressed firefly luciferase were isolated following transfection of the parental cells with luciferase cDNA. Each subline was passaged once or twice in vivo to enhance primary tumor growth and to increase metastasis. The resulting luciferase-expressing D3H1 and D3H2LN cells were analyzed for long-term bioluminescent stability, primary tumor growth, and distal metastasis to lymph nodes, lungs, bone and soft tissues by bioluminescent imaging. Cells were injected into the mammary fat pad of nude and nude-beige mice or were delivered systemically via intracardiac injection. Metastasis was also evaluated by ex vivo imaging and histologic analysis postmortem. RESULTS: The D3H1 and D3H2LN cell lines exhibited long-term stable luciferase expression for up to 4–6 months of accumulative tumor growth time in vivo. Bioluminescent imaging quantified primary mammary fat pad tumor development and detected early spontaneous lymph node metastasis in vivo. Increased frequency of spontaneous lymph node metastasis was observed with D3H2LN tumors as compared with D3H1 tumors. With postmortem ex vivo imaging, we detected additional lung micrometastasis in mice with D3H2LN mammary tumors. Subsequent histologic evaluation of tissue sections from lymph nodes and lung lobes confirmed spontaneous tumor metastasis at these sites. Following intracardiac injection of the MDA-MB-231-luc tumor cells, early metastasis to skeletal tissues, lymph nodes, brain and various visceral organs was detected. Weekly in vivo imaging data permitted longitudinal analysis of metastasis at multiple sites simultaneously. Ex vivo imaging data from sampled tissues verified both skeletal and multiple soft tissue tumor metastasis. CONCLUSION: This study characterized two new bioluminescent MDA-MB-231-luc human breast carcinoma cell lines with enhanced tumor growth and widespread metastasis in mice. Their application to current xenograft models of breast cancer offers rapid and highly sensitive detection options for preclinical assessment of anticancer therapies in vivo

Development of a Three-Dimensional In Vitro Model for Longitudinal Observation of Cell Behavior: Monitoring by Magnetic Resonance Imaging and Optical Imaging

Purpose: The aim of this study is the development of a three-dimensional multicellular spheroid cell culture model for the longitudinal comparative and large-scale screening of cancer cell proliferation with noninvasive molecular imaging techniques under controlled and quantifiable conditions. Procedures: The human glioblastoma cell line Gli36ΔEGFR was genetically modified to constitutively express the fluorescence protein mCherry, and additionally labeled with iron oxide nanoparticles for high-field MRI detection. The proliferation of aggregates was longitudinally monitored with fluorescence imaging and correlated with aggregate size by light microscopy, while MRI measurements served localization in 3D space. Irradiation with γ-rays was used to detect proliferational response. Results: Cell proliferation in the stationary three-dimensonal model can be observed over days with high accuracy. A linear relationship of fluorescence intensity with cell aggregate size was found, allowing absolute quantitation of cells in a wide range of cell amounts. Glioblastoma cells showed pronounced suppression of proliferation for several days following high-dose γ-irradiation. Conclusions: Through the combination of two-dimensional optical imaging and 3D MRI, the position of individual cell aggregates and their corresponding light emission can be detected. This allows an exact quantification of cell proliferation, with a focus on very small cell amounts (below 100 cells) using high resolution noninvasive techniques as a well-controlled basis for further cell transplantation studies

Noninvasive Monitoring of Placenta-Specific Transgene Expression by Bioluminescence Imaging

BACKGROUND: Placental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts. METHODOLOGY/PRINCIPAL FINDINGS: Zona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc) and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3). Animals were examined for Fluc expression by live bioluminescence imaging (BLI) at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm(2)/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages. CONCLUSIONS/SIGNIFICANCE: These results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early detection and quantitative analysis of gene expression throughout pregnancy by live BLI. This method may be useful for a wide range of applications involving trophoblast-specific gene manipulations in utero