Bioluminescence imaging on-chip platforms for non-invasive high-content bioimaging.

Incorporating non-invasive biosensing features in organ-on-chip models is of paramount importance for a wider implementation of these advanced in vitro microfluidic platforms. Optical biosensors, based on Bioluminescence Imaging (BLI), enable continuous, non-invasive, and in-situ imaging of cells, tissues or miniaturized organs without the drawbacks of conventional fluorescence imaging. Here, we report the first-of-its-kind integration and optimization of BLI in microfluidic chips, for non-invasive imaging of multiple biological readouts. The cell line HEK293T-GFP was engineered to express NanoLuc® luciferase under the control of a constitutive promoter and were cultured on-chip in 3D, in standard ECM-like hydrogels, to assess optimal cell detection conditions. Using real-time in-vitro dual-color microscopy, Bioluminescence (BL) and fluorescence (FL) were detectable using distinct imaging setups. Detection of the bioluminescent signals were observed at single cell resolution on-chip 20 min post-addition of Furimazine substrate and under perfusion. All hydrogels enabled BLI with higher signal-to-noise ratios as compared to fluorescence. For instance, agarose gels showed a ∼5-fold greater BL signal over background after injection of the substrate as compared to the FL signal. The use of BLI with microfluidic chip technologies opens up the potential for simultaneous in situ detection with continuous monitoring of multicolor cell reporters. Moreover, this can be achieved in a non-invasive manner. BL has great promise as a highly desirable biosensor for studying organ-on-chip platforms.

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

Introduction: The location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo. Methods: 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'). Results: 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. Conclusions: 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.

Advancing Tumor Microenvironment Research by Combining Organs-on-Chips and Biosensors.

Organs-on-chips are microfluidic tissue-engineered models that offer unprecedented dynamic control over cellular microenvironments, emulating key functional features of organs or tissues. Sensing technologies are increasingly becoming an essential part of such advanced model systems for real-time detection of cellular behavior and systemic-like events. The fast-developing field of organs-on-chips is accelerating the development of biosensors toward easier integration, thus smaller and less invasive, leading to enhanced access and detection of (patho-) physiological biomarkers. The outstanding combination of organs-on-chips and biosensors holds the promise to contribute to more effective treatments, and, importantly, improve the ability to detect and monitor several diseases at an earlier stage, which is particularly relevant for complex diseases such as cancer. Biosensors coupled with organs-on-chips are currently being devised not only to determine therapy effectiveness but also to identify emerging cancer biomarkers and targets. The ever-expanding use of imaging modalities for optical biosensors oriented toward on-chip applications is leading to less intrusive and more reliable detection of events both at the cellular and microenvironment levels. This chapter comprises an overview of hybrid approaches combining organs-on-chips and biosensors, focused on modeling and investigating solid tumors, and, in particular, the tumor microenvironment. Optical imaging modalities, specifically fluorescence and bioluminescence, will be also described, addressing the current limitations and future directions toward an even more seamless integration of these advanced technologies.

Near-Infrared Bioluminescence Imaging of Macrophage Sensors for Cancer Detection In Vivo.

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) in vivo, 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 in vivo 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.

Monitoring Immune Cell Function Through Optical Imaging: a Review Highlighting Transgenic Mouse Models.

Transgenic mouse models have facilitated research of human diseases and validation of therapeutic approaches. Inclusion of optical reporter genes (fluorescent or bioluminescent genes) in the targeting vectors used to develop such models makes in vivo imaging of cellular and molecular events possible, from the microscale to the macroscale. In particular, transgenic mouse models expressing optical reporter genes allowed accurately distinguishing immune cell types from trafficking in vivo using intravital microscopy or whole-body optical imaging. Besides lineage tracing and trafficking of different subsets of immune cells, the ability to monitor the function of immune cells is of pivotal importance for investigating the effects of immunotherapies against cancer. Here, we introduce the reader to state-of-the-art approaches to develop transgenics, optical imaging techniques, and several notable examples of transgenic mouse models developed for immunology research by critically highlighting the models that allow the following of immune cell function.

Dually Cross-Linked Core-Shell Structure Nanohydrogel with Redox-Responsive Degradability for Intracellular Delivery.

A redox-responsive nanocarrier is a promising strategy for the intracellular drug release because it protects the payload, prevents its undesirable leakage during extracellular transport, and favors site-specific drug delivery. In this study, we developed a novel redox responsive core-shell structure nanohydrogel prepared by a water in oil nanoemulsion method using two biocompatible synthetic polymers: vinyl sulfonated poly(N-(2-hydroxypropyl) methacrylamide mono/dilactate)-polyethylene glycol-poly(N-(2-hydroxypropyl) methacrylamide mono/dilactate) triblock copolymer, and thiolated hyaluronic acid. The influence on the nanohydrogel particle size and distribution of formulation parameters was investigated by a three-level full factorial design to optimize the preparation conditions. The surface and core-shell morphology of the nanohydrogel were observed by scanning electron microscope, transmission electron microscopy, and further confirmed by Fourier transform infrared spectroscopy and Raman spectroscopy from the standpoint of chemical composition. The redox-responsive biodegradability of the nanohydrogel in reducing environments was determined using glutathione as reducing agent. A nanohydrogel with particle size around 250 nm and polydispersity index around 0.1 is characterized by a thermosensitive shell which jellifies at body temperature and crosslinks at the interface of a redox-responsive hyaluronic acid core via the Michael addition reaction. The nanohydrogel showed good encapsulation efficiency for model macromolecules of different molecular weight (93% for cytochrome C, 47% for horseradish peroxidase, and 90% for bovine serum albumin), capacity to retain the peroxidase-like enzymatic activity (around 90%) of cytochrome C and horseradish peroxidase, and specific redox-responsive release behavior. Additionally, the nanohydrogel exhibited excellent cytocompatibility and internalization efficiency into macrophages. Therefore, the developed core-shell structure nanohydrogel can be considered a promising tool for the potential intracellular delivery of different pharmaceutical applications, including for cancer therapy.

Fluorinated PLGA-PEG-Mannose Nanoparticles for Tumor-Associated Macrophage Detection by Optical Imaging and MRI.

Tumor-associated macrophages (TAMs) promote cancer growth and metastasis, but their role in tumor development needs to be fully understood due to the dynamic changes of tumor microenvironment (TME). Here, we report an approach to visualize TAMs by optical imaging and by Fluorine-19 (19F) magnetic resonance imaging (MRI) that is largely applied to track immune cells in vivo. TAMs are targeted with PLGA-PEG-mannose nanoparticles (NPs) encapsulating perfluoro-15-crown-5-ether (PFCE) as MRI contrast agent. These particles are preferentially recognized and phagocytized by TAMs that overexpress the mannose receptor (MRC1/CD206). The PLGA-PEG-mannose NPs are not toxic and they were up-taken by macrophages as confirmed by in vitro confocal microscopy. At 48 h after intravenous injection of PLGA-PEG-mannose NPs, 4T1 xenograft mice were imaged and fluorine-19 nuclear magnetic resonance confirmed nanoparticle retention at the tumor site. Because of the lack of 19F background in the body, observed 19F signals are robust and exhibit an excellent degree of specificity. In vivo imaging of TAMs in the TME by 19F MRI opens the possibility for detection of cancer at earlier stage and for prompt therapeutic interventions in solid tumors.

Near-infrared bioluminescence imaging of two cell populations in living mice.

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).

Emerging tools for bioluminescence imaging.

Bioluminescence (BL) relies on the enzymatic reaction between luciferase, a substrate conventionally named luciferin, and various cofactors. BL imaging has become a widely used technique to interrogate gene expression and cell fate, both in small and large animal models of research. Recent developments include the generation of improved luciferase-luciferin systems for deeper and more sensitive imaging as well as new caged luciferins to report on enzymatic activity and other intracellular functions. Here, we critically evaluate the emerging tools for BL imaging aiming to provide the reader with an updated compendium of the latest developments (2018-2020) and their notable applications.

Evaluation of NanoLuc substrates for bioluminescence imaging of transferred cells in mice.

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.

Theranostic Design of Angiopep-2 Conjugated Hyaluronic Acid Nanoparticles (Thera-ANG-cHANPs) for Dual Targeting and Boosted Imaging of Glioma Cells.

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 T1 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.

Development of a New Hyaluronic Acid Based Redox-Responsive Nanohydrogel for the Encapsulation of Oncolytic Viruses for Cancer Immunotherapy.

Oncolytic viruses (OVs) are emerging as promising and potential anti-cancer therapeutic agents, not only able to kill cancer cells directly by selective intracellular viral replication, but also to promote an immune response against tumor. Unfortunately, the bioavailability under systemic administration of OVs is limited because of undesired inactivation caused by host immune system and neutralizing antibodies in the bloodstream. To address this issue, a novel hyaluronic acid based redox responsive nanohydrogel was developed in this study as delivery system for OVs, with the aim to protect the OVs following systemic administration. The nanohydrogel was formulated by water in oil (W/O) nanoemulsion method and cross-linked by disulfide bonds derived from the thiol groups of synthesized thiolated hyaluronic acid. One DNA OV Ad[I/PPT-E1A] and one RNA OV Rigvir® ECHO-7 were encapsulated into the developed nanohydrogel, respectively, in view of their potential of immunovirotherapy to treat cancers. The nanohydrogels showed particle size of approximately 300-400 nm and negative zeta potential of around -13 mV by dynamic light scattering (DLS). A uniform spherical shape of the nanohydrogel was observed under the scanning electron microscope (SEM) and transmission electron microscope (TEM), especially, the successfully loading of OV into nanohydrogel was revealed by TEM. The crosslinking between the hyaluronic acid chains was confirmed by the appearance of new peak assigned to disulfide bond in Raman spectrum. Furthermore, the redox responsive ability of the nanohydrogel was determined by incubating the nanohydrogel into phosphate buffered saline (PBS) pH 7.4 with 10 µM or 10 mM glutathione at 37 °C which stimulate the normal physiological environment (extracellular) or reductive environment (intracellular or tumoral). The relative turbidity of the sample was real time monitored by DLS which indicated that the nanohydrogel could rapidly degrade within 10 h in the reductive environment due to the cleavage of disulfide bonds, while maintaining the stability in the normal physiological environment after 5 days. Additionally, in vitro cytotoxicity assays demonstrated a good oncolytic activity of OVs-loaded nanohydrogel against the specific cancer cell lines. Overall, the results indicated that the developed nanohydrogel is a delivery system appropriate for viral drugs, due to its hydrophilic and porous nature, and also thanks to its capacity to maintain the stability and activity of encapsulated viruses. Thus, nanohydrogel can be considered as a promising candidate carrier for systemic administration of oncolytic immunovirotherapy.

Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging.

For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH2-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate.

Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging.

PURPOSE: Currently, a variety of red and green beetle luciferase variants are available for bioluminescence imaging (BLI). In addition, new luciferin analogues providing longer wavelength luminescence have been developed that show promise for improved deep tissue imaging. However, a detailed assessment of these analogues (e.g., Akalumine-HCl, CycLuc1, and amino naphthyl luciferin (NH2-NpLH2)) combined with state of the art luciferases has not been performed. The aim of this study was to evaluate for the first time the in vivo brightness and spectral characteristics of firefly (Luc2), click beetle green (CBG99), click beetle red 2 (CBR2), and Akaluc luciferases when paired with different D-luciferin (D-LH2) analogues in vivo. PROCEDURES: Transduced human embryonic kidney (HEK 293T) cells expressing individual luciferases were analyzed both in vitro and in mice (via subcutaneous injection). Following introduction of the luciferins to cells or animals, the resulting bioluminescence signal and photon emission spectrum were acquired using a sensitive charge-coupled device (CCD) camera equipped with a series of band pass filters and spectral unmixing software. RESULTS: Our in vivo analysis resulted in four primary findings: (1) the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was D-luciferin; (2) the spectra for Luc2 and CBR2 were shifted to a longer wavelength when Akalumine-HCl was the substrate; (3) CBR2 gave the brightest signal with the near-infrared substrate, NH2-NpLH2; and (4) Akaluc was brighter when paired with either CycLuc1 or Akalumine-HCl when paired with D-LH2. CONCLUSION: We believe that the experimental results described here should provide valuable guidance to end users for choosing the correct luciferin/luciferase pairs for a variety of BLI applications.

NanoBiT System and Hydrofurimazine for Optimized Detection of Viral Infection in Mice-A Novel in Vivo Imaging Platform.

Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models.

Active Nano-targeting of Macrophages.

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.

A Dual-Color Bioluminescence Reporter Mouse for Simultaneous in vivo Imaging of T Cell Localization and Function.

Non-invasive imaging technologies to visualize the location and functionality of T cells are of great value in immunology. Here, we describe the design and generation of a transgenic mouse in which all T cells constitutively express green-emitting click-beetle luciferase (CBG99) while expression of the red-emitting firefly luciferase (PpyRE9) is induced by Nuclear Factor of Activated T cells (NFAT) such as during T cell activation, which allows multicolor bioluminescence imaging of T cell location and function. This dual-luciferase mouse, which we named TbiLuc, showed high constitutive luciferase expression in lymphoid organs such as lymph nodes and the spleen. Ex vivo purified CD8+ and CD4+ T cells both constitutively expressed luciferase, whereas B cells showed no detectable signal. We cross-bred TbiLuc mice to T cell receptor-transgenic OT-I mice to obtain luciferase-expressing naïve CD8+ T cells with defined antigen-specificity. TbiLuc*OT-I T cells showed a fully antigen-specific induction of the T cell activation-dependent luciferase. In vaccinated mice, we visualized T cell localization and activation in vaccine-draining lymph nodes with high sensitivity using two distinct luciferase substrates, D-luciferin and CycLuc1, of which the latter specifically reacts with the PpyRE9 enzyme. This dual-luciferase T cell reporter mouse can be applied in many experimental models studying the location and functional state of T cells.