The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is ...high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.
Active Nano-targeting of Macrophages Gaspar, Natasa; Zambito, Giorgia; Löwik, Clemens M W G ...
Current pharmaceutical design,
01/2019, Letnik:
25, Številka:
17
Journal Article
Recenzirano
Macrophages play a role in almost every disease such as cancer, infections, injuries, metabolic and inflammatory diseases and are becoming an attractive therapeutic target. However, understanding ...macrophage diversity, tissue distribution and plasticity will help in defining precise targeting strategies and effective therapies. Active targeting of macrophages using nanoparticles for therapeutic purposes is still at its infancy but holds promises since macrophages have shown high specific uptake of nanoparticles. Here, we highlight recent progress in active nanotechnology-based systems gaining pivotal roles to target diverse macrophage subsets in diseased tissues.
The unmet demand for the development of advanced in vitro models that emulate key features of human (patho‐)physiology and organ or tissue functionality led to the emergence of organ‐on‐a‐chip ...screening platforms. This biomimetic microtechnology enables accurate prediction of human responses, even at the multitissue or organ interaction level. With these advances, the necessity of incorporating biosensing within these platform becomes increasingly evident, to ultimately allow in situ, noninvasive, monitoring of cells, tissues and organs’ behavior. Seamlessly integrated biosensors on‐chip also offer the prospect of uninterrupted and fully automated analysis. The continuous search for biosensors that not only avoid disruptive analyses, but are also highly sensitive and permit instant in situ evaluation, steered further developments of optical biosensors including bioluminescence (BL). BL is particularly suitable to interrogate biological systems at the multiparameter level and it is well matched to address the intrinsic biological systems’ heterogeneity due to its inherent high signal to background ratio, to which the demand of simple measurement equipment can be added. Moreover, BL can be effortlessly integrated into any available organ‐on‐a‐chip platform and provides unique groundbreaking means for online monitoring, at the cellular level, and/or detection of cell secreted/excreted compounds. This review depicts recent advances in biosensors, with particular focus on BL. We highlight current challenges and future directions, aimed at stimulating the application of BL to interface organ‐on‐a‐chip systems, leading to the exponential advancement of both fields.
The integration of organ‐on‐a‐chip models with biosensors, in particular based on bioluminescence (BL), uniquely enables online, real‐time and non‐invasive monitoring of cellular events, with an unprecedent balance of sensitivity of detection, multiplexing, cost effectiveness and easiness of use. Here, we highlight the potential of combining BL strategies with organ‐on‐a‐chip systems, focusing on applications and anticipated advancements, expected increase the speed and efficacy of sensing key biological events. In turn, such innovative complementary technologies hold the promise to improve our understanding of human diseases, drug development and patient‐specific therapy screening.
NanoLuc luciferase recently gained popularity due to its small size and superior bioluminescence performance. For in vivo imaging applications, NanoLuc has been limited by its substrate furimazine, ...which has low solubility and bioavailability. Herein, we compared the performances of recently reported NanoLuc luciferase substrates for in vivo imaging in mice. Two substrates with improved aqueous solubility, hydrofurimazine and fluorofurimazine, were evaluated along with three stabilized O-acetylated furimazine analogues, the hikarazines. All 5 analogues, when tested in vitro, displayed greater signal intensity and reaction duration, in comparison to the standard NanoLuc substrate, furimazine. The two best-performing analogues from the in vitro study were selected for further in vivo testing. The NanoLuc/fluorofurimazine pair demonstrated the highest bioluminescence intensity, post intravenous administration. It was found to be around 9-fold brighter compared to the NanoLuc/furimazine and 11-fold more intense than the NanoLuc/hikarazine-003 pair, with an average of 3-fold higher light emission when the substrate was injected intraperitoneally, in a subcutaneous model. Excitingly, despite the fact that NanoLuc/fluorofurimazine emits mostly blue light, we prove that cells trapped in mice lungs vasculature could be visualised via the NanoLuc/fluorofurimazine pair and compare the results to the AkaLuc/AkaLumine system. Therefore, among the tested analogues, fluorofurimazine enables higher substrate loading and improved optical imaging sensitivity in small animals, upgrading the use of NanoLuc derived bioluminescent systems for deep tissue imaging.
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•Hikarazine-003 and fluorofurimazine enable higher signal emission than furimazine for bioluminescence imaging.•Fluorofurimazine enables higher substrate loading and improved optical imaging sensitivity in mice.•Fluorofurimazine enable deep tissue imaging of NanoLuc expressing transferred cells.
The efficacy of immunotherapy against advanced cancer may be improved by combination strategies. Photodynamic therapy (PDT) is a local tumor ablation method based on localized activation of a ...photosensitizer, leading to oxygen radical-induced tumor cell death. PDT can enhance antitumor immune responses by release of antigen and danger signals, supporting combination protocols of PDT with immunotherapy.
We investigated the local and systemic immune effects of PDT after treatment of established tumors. In two independent aggressive mouse tumor models, TC-1 and RMA, we combined PDT with therapeutic vaccination using synthetic long peptides (SLP) containing epitopes from tumor antigens.
PDT of established tumors using the photosensitizer Bremachlorin resulted in significant delay of tumor outgrowth. Combination treatment of PDT with therapeutic SLP vaccination cured one third of mice. Importantly, all cured mice were fully protected against subsequent tumor rechallenge, and combination treatment of primary tumors led to eradication of distant secondary tumors, indicating the induction of a systemic antitumor immune response. Indeed, PDT by itself induced a significant CD8(+) T-cell response against the tumor, which was increased when combined with SLP vaccination and essential for the therapeutic effect of combination therapy.
We show that immunotherapy can be efficiently combined with PDT to eradicate established tumors, based on strong local tumor ablation and the induction of a robust systemic immune response. These results suggest combination of active immunotherapy with tumor ablation by PDT as a feasible novel treatment strategy for advanced cancer.
Multicolor bioluminescence imaging using near-infrared emitting luciferases is an attractive application to detect two cell populations within one animal model. Herein, we describe how to distinguish ...dual-color bioluminescent signals co-localized in the same compartment. We tested CBG2 click beetle (λ = 660 nm) and CBR2 click beetle (λ = 730 nm) luciferases paired with NH2-NpLH2 luciferin. Following a spectral unmixing algorithm, single spectral contributions can be resolved and quantified, enabling the visualization of multiple cell types in deep tissue by injection of a single substrate.
For complete details on the use and execution of this protocol, please refer to Zambito et al. (2020).
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•A protocol for dual-color bioluminescence imaging in deep tissue is provided•A step-by-step spectral unmixing tool is applied for spectral properties of luciferases•Co-localized green and red signals can be separated and quantified in vivo
Multicolor bioluminescence imaging using near-infrared emitting luciferases is an attractive application to detect two cell populations within one animal model. Herein, we describe how to distinguish dual-color bioluminescent signals co-localized in the same compartment. We tested CBG2 click beetle (λ = 660 nm) and CBR2 click beetle (λ = 730 nm) luciferases paired with NH2-NpLH2 luciferin. Following a spectral unmixing algorithm, single spectral contributions can be resolved and quantified, enabling the visualization of multiple cell types in deep tissue by injection of a single substrate.
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 color-coupled 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.
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.
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, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.
Melanoma is an aggressive type of skin cancer with a poor prognosis after it gets metastasized. The early detection of malignant melanoma is critical for effective therapy. Because melanoma often ...resembles moles, routine skin check-up may help for timely identification of suspicious areas. Recently, it has been shown that the interplay of melanoma cells with the immune system can help develop efficient therapeutic strategies. Here, we leveraged engineered macrophages (BMC2) as cell-based sensors for metastatic melanoma. To perform dual-color bioluminescence imaging (BLI)
, macrophages were engineered to express a green click beetle luciferase (CBG2) and a near-infrared fluorescent dye (DiR), and B16F10 melanoma cells were instead engineered to express a near-infrared click beetle luciferase (CBR2). Using real-time
dual-color BLI and near-infrared fluorescence (FL) imaging, we could demonstrate that macrophages were able to sense and substantially accumulate in subcutaneous and metastatic melanoma tissues at 72 h after systemic injections. Together, we showed the potentiality to use optical imaging technologies to track circulating macrophages for the non-invasive detection of metastatic melanoma.
Glioblastoma multiforme (GBM) has a mean survival of only 15 months. Tumour heterogeneity and blood-brain barrier (BBB) mainly hinder the transport of active agents, leading to late diagnosis, ...ineffective therapy and inaccurate follow-up. The use of hydrogel nanoparticles, particularly hyaluronic acid as naturally occurring polymer of the extracellular matrix (ECM), has great potential in improving the transport of drug molecules and, furthermore, in facilitatating the early diagnosis by the effect of hydrodenticity enabling the T
boosting of Gadolinium chelates for MRI. Here, crosslinked hyaluronic acid nanoparticles encapsulating gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) and the chemotherapeutic agent irinotecan (Thera-cHANPs) are proposed as theranostic nanovectors, with improved MRI capacities. Irinotecan was selected since currently repurposed as an alternative compound to the poorly effective temozolomide (TMZ), generally approved as the gold standard in GBM clinical care. Also, active crossing and targeting are achieved by theranostic cHANPs decorated with angiopep-2 (Thera-ANG-cHANPs), a dual-targeting peptide interacting with low density lipoprotein receptor related protein-1(LRP-1) receptors overexpressed by both endothelial cells of the BBB and glioma cells. Results showed preserving the hydrodenticity effect in the advanced formulation and internalization by the active peptide-mediated uptake of Thera-cHANPs in U87 and GS-102 cells. Moreover, Thera-ANG-cHANPs proved to reduce ironotecan time response, showing a significant cytotoxic effect in 24 h instead of 48 h.
The location of T-cells during tumor progression and treatment provides crucial information in predicting the response
.
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
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.
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