In vivo bioluminescence imaging of the intracerebral fibroin-controlled AAV-α-synuclein diffusion for monitoring the central nervous system and peripheral expression.

Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.

CRISPR editing to mimic porphyria combined with light: A new preclinical approach for prostate cancer.

Thanks to its very high genome-editing efficiency, CRISPR-Cas9 technology could be a promising anticancer weapon. Clinical trials using CRISPR-Cas9 nuclease to ex vivo edit and alter immune cells are ongoing. However, to date, this strategy still has not been applied in clinical practice to directly target cancer cells. Targeting a canonical metabolic pathway essential to good functioning of cells without potential escape would represent an attractive strategy. We propose to mimic a genetic metabolic disorder in cancer cells to weaken cancer cells, independent of their genomic abnormalities. Mutations affecting the heme biosynthesis pathway are responsible for porphyria, and most of them are characterized by an accumulation of toxic photoreactive porphyrins. This study aimed to mimic porphyria by using CRISPR-Cas9 to inactivate UROS, leading to porphyrin accumulation in a prostate cancer model. Prostate cancer is the leading cancer in men and has a high mortality rate despite therapeutic progress, with a primary tumor accessible to light. By combining light with gene therapy, we obtained high efficiency in vitro and in vivo, with considerable improvement in the survival of mice. Finally, we achieved the preclinical proof-of-principle of performing cancer CRISPR gene therapy.

Targeting metastasis-initiating cancer stem cells in gastric cancer with leukaemia inhibitory factor.

Gastric cancer's (GC) bad prognosis is usually associated with metastatic spread. Invasive cancer stem cells (CSC) are considered to be the seed of GC metastasis and not all CSCs are able to initiate metastasis. Targeting these aggressive metastasis-initiating CSC (MIC) is thus vital. Leukaemia inhibitory factor (LIF) is hereby used to target Hippo pathway oncogenic members, found to be induced in GC and associated with CSC features. LIF-treated GC cell lines, patient-derived xenograft (PDX) cells and/or CSC tumourspheres underwent transcriptomics, laser microdissection-associated proteomics, 2D and 3D invasion assays and in vivo xenograft in mice blood circulation. LIFR expression was analysed on tissue microarrays from GC patients and in silico from public databases. LIF-treated cells, especially CSC, presented decreased epithelial to mesenchymal transition (EMT) phenotype and invasion capacity in vitro, and lower metastasis initiation ability in vivo. These effects involved both the Hippo and Jak/Stat pathways. Finally, GC's high LIFR expression was associated with better clinical outcomes in patients. LIF treatment could thus represent a targeted anti-CSC strategy to fight against metastatic GC, and LIFR detection in primary tumours could constitute a potential new prognosis marker in this disease.

Inhibition of proprotein convertases activity results in repressed stemness and invasiveness of cancer stem cells in gastric cancer.

BACKGROUND: Gastric cancer (GC), the fourth leading cause of cancer-related death worldwide, with most deaths caused by advanced and metastatic disease, has limited curative options. Here, we revealed the importance of proprotein convertases (PCs) in the malignant and metastatic potential of GC cells through the regulation of the YAP/TAZ/TEAD pathway and epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSC). METHODS: The general PCs inhibitor, decanoyl-RVKR-chloromethyl-ketone (CMK), was used to repress PCs activity in CSCs of various GC cell lines. Their tumorigenic properties, drug resistance, YAP/TAZ/TEAD pathway activity, and invasive properties were then investigated in vitro, and their metastatic properties were explored in a mouse xenograft model. The prognostic value of PCs in GC patients was also explored in molecular databases of GC. RESULTS: Inhibition of PCs activity in CSCs in all GC cell lines reduced tumorsphere formation and growth, drug efflux, EMT phenotype, and invasive properties that are associated with repressed YAP/TAZ/TEAD pathway activity in vitro. In vivo, PCs' inhibition in GC cells reduced their metastatic spread. Molecular analysis of tumors from GC patients has highlighted the prognostic value of PCs. CONCLUSIONS: PCs are overexpressed in GC and associated with poor prognosis. PCs are involved in the malignant and metastatic potential of CSCs via the regulation of EMT, the YAP/TAZ/TEAD oncogenic pathway, and their stemness and invasive properties. Their repression represents a new strategy to target CSCs and impair metastatic spreading in GC.

The role of tumor model in magnetic targeting of magnetosomes and ultramagnetic liposomes.

The combined passive and active targeting of tumoral tissue remains an active and relevant cancer research field. Here, we exploit the properties of two highly magnetic nanomaterials, magnetosomes and ultramagnetic liposomes, in order to magnetically target prostate adenocarcinoma tumors, implanted orthotopically or subcutaneously, to take into account the role of tumor vascularization in the targeting efficiency. Analysis of organ biodistribution in vivo revealed that, for all conditions, both nanomaterials accumulate mostly in the liver and spleen, with an overall low tumor retention. However, both nanomaterials were more readily identified in orthotopic tumors, reflecting their higher tumor vascularization. Additionally, a 2- and 3-fold increase in nanomaterial accumulation was achieved with magnetic targeting. In summary, ultramagnetic nanomaterials show promise mostly in the targeting of highly-vascularized orthotopic murine tumor models.

New insights into detecting alizarin from autofluorescence in marked glass eels.

Alizarin detection in fish fins is extensively employed because it is easy to use. However, in eels, the eelGFP fluorescent protein may impede the detection of the fluorescent markers in the eel tissues. The study tests the effectiveness of three of the most up-to-date alizarin-detecting technologies on the living body and fins of European glass eels (Anguilla anguilla L.). The findings demonstrated that the control group had a high autofluorescence at alizarin and eelGFP maxima bands. With fluorescence reflectance imaging (FRI), the eel living body autofluorescence impeded the detection of the marked eels. In contrast with experimental excitation-emission-matrix (EEM) fluorescence analyses, 99% of the marked eels were correctly assigned to their group from fluorescence analyses of their fin cellular contents. With epifluorometry (EPI), 100% of the marked eels were detected with the caudal fin tips when excited at 450-490 nm wavelengths due to a weaker autofluorescence signal. EEM and FRI assays unveiled an average fluorescence quenching 60% and 44% of the marked group respectively, in the alizarin and eelGFP maxima bands. The fluorescence quenching observed is discussed. Results will benefit experimental design by examining autofluorescence effects on mark detection and the development of non-invasive detection methods in this critically endangered species.

In Vivo Follow-Up of Gene Inhibition in Solid Tumors Using Peptide-Based Nanoparticles for siRNA Delivery.

Small interfering RNA (siRNA) exhibits a high degree of specificity for targeting selected genes. They are efficient on cells in vitro, but in vivo siRNA therapy remains a challenge for solid tumor treatment as siRNAs display difficulty reaching their intracellular target. The present study was designed to show the in vivo efficiency of a new peptide (WRAP5), able to form peptide-based nanoparticles (PBN) that can deliver siRNA to cancer cells in solid tumors. WRAP5:siRNA nanoparticles targeting firefly luciferase (Fluc) were formulated and assayed on Fluc-expressing U87 glioblastoma cells. The mode of action of WRAP5:siRNA by RNA interference was first confirmed in vitro and then investigated in vivo using a combination of bioluminescent reporter genes. Finally, histological analyses were performed to elucidate the cell specificity of this PBN in the context of brain tumors. In vitro and in vivo results showed efficient knock-down of Fluc expression with no toxicity. WRAP5:siFluc remained in the tumor for at least 10 days in vivo. Messenger RNA (mRNA) analyses indicated a specific decrease in Fluc mRNA without affecting tumor growth. Histological studies identified PBN accumulation in the cytoplasm of tumor cells but also in glial and neuronal cells. Through in vivo molecular imaging, our findings established the proof of concept for specific gene silencing in solid tumors. The evidence generated could be translated into therapy for any specific gene in different types of tumors without cell type specificity but with high molecular specificity.

Multimodal optical contrast agents as new tools for monitoring and tuning nanoemulsion internalisation into cancer cells. From live cell imaging to in vivo imaging of tumours.

Tailor-made NIR emitting dyes were designed as multimodal optical probes. These asymmetric amphiphilic compounds show combined intense absorption in the visible region, NIR fluorescence emission, high two-photon absorption in the NIR (with the maximum located around 1000 nm) as well as large Stokes' shift values and second-harmonic generation ability. Thanks to their structure, high loading into nanoemulsions (NEs) could be achieved leading to very high one- and two-photon brightness. These dyes were demonstrated to act as multimodal contrast agents able to generate different optical modalities of interest for bioimaging. Indeed, the uptake and carrier behaviour of the dye-loaded NEs into cancer cells could be monitored by simultaneous two-photon fluorescence and second-harmonic generation optical imaging. Multimodal imaging provided deep insight into the mechanism and kinetics of dye internalisation. Quite interestingly, the nature of the dyes was also found to influence both the kinetics of endocytosis and the internalisation pathways in glioblastoma cancer cells. By modulating the charge distribution within the dyes, the NEs can be tuned to escape lysosomes and enter the mitochondria. Moreover, surface functionalization with PEG macromolecules was realized to yield stealth NIRF-NEs which could be used for in vivo NIRF imaging of subcutaneous tumours in mice.

Imaging of conditional gene silencing in vivo using a bioluminescence-based method with thermo-inducible microRNAs.

RNA interference (RNAi)-based gene therapy has great potential in cancer and infectious disease treatment to correct abnormal up-regulation of gene expression. We show a new original method uses synthetic microRNAs combined with a thermo-inducible promoter to reduce specific gene expression. The targeted gene is the luciferase firefly reporter gene overexpressed in a subcutaneous tumor which allows the RNAi monitoring by bioluminescence imaging (BLI). The inducible inhibition was first demonstrated in vitro using genetically modified cells lines and then in vivo using the corresponding xenograft model in mice. Achieving spatio-temporal control, we demonstrate the feasibility to induce, in vivo, a specific gene inhibition on demand. Future applications of this RNAi-based gene therapy, which can be restricted to pathological tissue, would offer wide-ranging potential for disease treatment.

In Vivo Imaging of Prostate Cancer Tumors and Metastasis Using Non-Specific Fluorescent Nanoparticles in Mice.

With the growing interest in the use of nanoparticles (NPs) in nanomedicine, there is a crucial need for imaging and targeted therapies to determine NP distribution in the body after systemic administration, and to achieve strong accumulation in tumors with low background in other tissues. Accumulation of NPs in tumors results from different mechanisms, and appears extremely heterogeneous in mice models and rather limited in humans. Developing new tumor models in mice, with their low spontaneous NP accumulation, is thus necessary for screening imaging probes and for testing new targeting strategies. In the present work, accumulation of LipImageTM 815, a non-specific nanosized fluorescent imaging agent, was compared in subcutaneous, orthotopic and metastatic tumors of RM1 cells (murine prostate cancer cell line) by in vivo and ex vivo fluorescence imaging techniques. LipImageTM 815 mainly accumulated in liver at 24 h but also in orthotopic tumors. Limited accumulation occurred in subcutaneous tumors, and very low fluorescence was detected in metastasis. Altogether, these different tumor models in mice offered a wide range of NP accumulation levels, and a panel of in vivo models that may be useful to further challenge NP targeting properties.

A retro-inverso cell-penetrating peptide for siRNA delivery.

BACKGROUND: Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. RESULTS: Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. CONCLUSIONS: This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins). Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.

PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo.

Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro-inverso amphipathic RICK peptide as novel non-covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by self-assembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties. A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤20%) of the NPs did not influence their cellular internalization capacity as well as their knock-down specificity (over-expressed or endogenous system) in vitro. Because the behavior of PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labeled NPs injected at the one-cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after administration to mice. After an intra-cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG-RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation constitutes a modular, easy-to-handle drug delivery system which could be adapted to other types of functional moieties to develop safe and biocompatible delivery systems for the clinical application of RNAi-based cancer therapeutics.

In Vivo Imaging of Local Gene Expression Induced by Magnetic Hyperthermia.

The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release.

Impact of surface grafting density of PEG macromolecules on dually fluorescent silica nanoparticles used for the in vivo imaging of subcutaneous tumors.

BACKGROUND: In the context of systematically administered nanomedicines, the physicochemistry of NP surfaces must be controlled as a prerequisite to improve blood circulation time, and passive and active targeting. In particular, there is a real need to develop NP stealth and labelling for both in vivo and microscopic fluorescence imaging in a mice model. METHODS: We have synthesized NIR/red dually fluorescent silica nanoparticles of 19nm covalently covered by a PEG layer of different grafting density in the brush conformational regime by using a reductive amination reaction. These particles were characterized by TEM, DRIFT, DLS, TGA, ζ potential measurements, UV-vis and fluorescence spectroscopy. Prostate tumors were generated in mice by subcutaneous injection of RM1-CMV-Fluc cells. Tumor growth was monitored by BLI after a D-luciferin injection. Four samples of PEGylated fluorescent NPs were individually intravenously injected into 6 mice (N=6, total 24 mice). Nanoparticle distribution was investigated using in vivo fluorescence reflectance imaging (FRI) over 48h and microscopy imaging was employed to localize the NPs within tumors in vitro. RESULTS: Fluorescent NP accumulation, due to the enhanced permeability and retention (EPR) effect, increases gradually as a function of increased PEG surface grafting density with a huge difference observed for the highest density grafting. For the highest grafting density, a blood circulation time of up to 24h was observed with a strong reduction in uptake by the liver. In vivo experimental results suggest that the biodistribution of NPs is very sensitive to slight variations in surface grafting density when the NPs present a high curvature radius. CONCLUSION: This study underlines the need to compensate a high curvature radius with a PEG-saturated NP surface to improve blood circulation and accumulation within tumors through the EPR effect. Dually fluorescent NPs PEGylated to saturation display physical properties useful for assessing the susceptibility of tumors to the EPR effect. GENERAL SIGNIFICANCE: Control of the physicochemical features of nanoparticle surfaces to improve blood circulation times and monitoring of the EPR effect. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography.

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT®). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors.

In vivo imaging of prostate cancer using an anti-PSMA scFv fragment as a probe.

We aimed to evaluate a fluorescent-labeled single chain variable fragment (scFv) of the anti-PSMA antibody as a specific probe for the detection of prostate cancer by in vivo fluorescence imaging. An orthotopic model of prostate cancer was generated by injecting LNCaP cells into the prostate lobe. ScFvD2B, a high affinity anti-PSMA antibody fragment, was labeled using a near-infrared fluorophore to generate a specific imaging probe (X770-scFvD2B). PSMA-unrelated scFv-X770 was used as a control. Probes were injected intravenously into mice with prostate tumors and fluorescence was monitored in vivo by fluorescence molecular tomography (FMT). In vitro assays showed that X770-scFvD2B specifically bound to PSMA and was internalized in PSMA-expressing LNCaP cells. After intravenous injection, X770-scFvD2B was detected in vivo by FMT in the prostate region. On excised prostates the scFv probe co-localized with the cancer cells and was found in PSMA-expressing cells. The PSMA-unrelated scFv used as a control did not label the prostate cancer cells. Our data demonstrate that scFvD2B is a high affinity contrast agent for in vivo detection of PSMA-expressing cells in the prostate. NIR-labeled scFvD2B could thus be further developed as a clinical probe for imaging-guided targeted biopsies.

Detection of Brain Tumors and Systemic Metastases Using NanoLuc and Fluc for Dual Reporter Imaging.

PURPOSE: Bioluminescence imaging (BLI) is a technique with a low background noise and high sensitivity which is widely used in mice models in oncology. We aimed to assess BLI efficiency of the new luciferase NanoLuc (Nluc) for glioblastoma cell lines and tumors, including for dual reporter applications of deep brain tumors and systemic metastasis when combined with firefly luciferase (Fluc). PROCEDURES: U87 cells were genetically modified for constitutive production of either Nluc, Fluc, or both and assayed for luciferase activity and BLI on cell lysates, living cells, subcutaneous tumors, brain tumors, and systemic metastases. RESULTS: In vitro, light production by Nluc activity is higher than Fluc. In vivo, Nluc allows for tumor detection including for deep brain tumors and systemic metastases. CONCLUSIONS: Nluc appears to be a useful tool to combine with Fluc for dual imaging in vivo using bioluminescence, allowing for the detection of distinct events in deep tissues within the same organism.

Spatiotemporal control of gene expression in bone-marrow derived cells of the tumor microenvironment induced by MRI guided focused ultrasound.

The tumor microenvironment is an interesting target for anticancer therapies but modifying this compartment is challenging. Here, we demonstrate the feasibility of a gene therapy strategy that combined targeting to bone marrow-derived tumor microenvironment using genetically modified bone-marrow derived cells and control of transgene expression by local hyperthermia through a thermo-inducible promoter. Chimera were obtained by engraftment of bone marrow from transgenic mice expressing reporter genes under transcriptional control of heat shock promoter and inoculated sub-cutaneously with tumors cells. Heat shocks were applied at the tumor site using a water bath or magnetic resonance guided high intensity focused ultrasound device. Reporter gene expression was followed by bioluminescence and fluorescence imaging and immunohistochemistry. Bone marrow-derived cells expressing reporter genes were identified to be mainly tumor-associated macrophages. We thus provide the proof of concept for a gene therapy strategy that allows for spatiotemporal control of transgenes expression by macrophages targeted to the tumor microenvironment.

Dual-reporter in vivo imaging of transient and inducible heat-shock promoter activation.

Gene promoter activity can be studied in vivo by molecular imaging methods using reporter gene technology. Transcription of the reporter and the reported genes occurs simultaneously. However, imaging depends on reporter protein translation, stability, and cellular fate that may differ among the various proteins. A double transgenic mouse strain expressing the firefly luciferase (lucF) and fluorescent mPlum protein under the transcriptional control of the thermo-inducible heat-shock protein (Hspa1b) promoter was generated allowing to follow up the reporter proteins by different and complementary in vivo imaging technologies. These mice were used for in vivo imaging by bioluminescence and epi fluorescence reflectance imaging (BLI & FRI) and as a source of embryonic fibroblast (MEF) for in vitro approaches. LucF, mPlum and endogenous Hsp70 mRNAs were transcribed simultaneously. The increase in mRNA was transient, peaking at 3 h and then returning to the basal level about 6 h after the thermal stimulations. The bioluminescent signal was transient and initiated with a 3 h delay versus mRNA expression. The onset of mPlum fluorescence was more delayed, increasing slowly up to 30 h after heat-shock and remaining for several days. This mouse allows for both bioluminescence imaging (BLI) and fluorescence reflectance imaging (FRI) of Hsp70 promoter activation showing an early and transient lucF activity and a retrospective and persistent mPlum fluorescence. This transgenic mouse will allow following the transient local induction of Hsp-70 promoter beyond its induction time-frame and relate into subsequent dynamic biological effects of the heat-shock response.