New technologies for improved relevance in miRNA research.

After a number of years of research in the field of miRNA, the robustness and biological relevance of many published articles is increasingly being questioned. We propose the use of new RNA-seq approaches, genome editing technologies, and updated public databases to improve the quality, reliability, and relevance of published data.

Identification of oncolytic vaccinia restriction factors in canine high-grade mammary tumor cells using single-cell transcriptomics.

Mammary carcinoma, including triple-negative breast carcinomas (TNBC) are tumor-types for which human and canine pathologies are closely related at the molecular level. The efficacy of an oncolytic vaccinia virus (VV) was compared in low-passage primary carcinoma cells from TNBC versus non-TNBC. Non-TNBC cells were 28 fold more sensitive to VV than TNBC cells in which VV replication is impaired. Single-cell RNA-seq performed on two different TNBC cell samples, infected or not with VV, highlighted three distinct populations: naïve cells, bystander cells, defined as cells exposed to the virus but not infected and infected cells. The transcriptomes of these three populations showed striking variations in the modulation of pathways regulated by cytokines and growth factors. We hypothesized that the pool of genes expressed in the bystander populations was enriched in antiviral genes. Bioinformatic analysis suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. In addition, we demonstrated experimentally that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. This information could be used to design new generations of oncolytic poxviruses. Beyond the field of gene therapy, this study demonstrates that single-cell transcriptomics can be used to identify cellular factors influencing viral replication.

Mesenchymal Stem Cells Delivery in Individuals with Different Pathologies: Multimodal Tracking, Safety and Future Applications.

Due to their ease of isolation and their properties, mesenchymal stem cells (MSCs) have been widely investigated. MSCs have been proved capable of migration towards areas of inflammation, including tumors. Therefore, they have been suggested as vectors to carry therapies, specifically to neoplasias. As most of the individuals joining clinical trials that use MSCs for cancer and other pathologies are carefully recruited and do not suffer from other diseases, here we decided to study the safety and application of iv-injected MSCs in animals simultaneously induced with different inflammatory pathologies (diabetes, wound healing and tumors). We studied this by in vitro and in vivo approaches using different gene reporters (GFP, hNIS, and f-Luc) and non-invasive techniques (PET, BLI, or fluorescence). Our results found that MSCs reached different organs depending on the previously induced pathology. Moreover, we evaluated the property of MSCs to target tumors as vectors to deliver adenoviruses, including the interaction between tumor microenvironment and MSCs on their arrival. Mechanisms such as transdifferentiation, MSC fusion with cells, or paracrine processes after MSCs homing were studied, increasing the knowledge and safety of this new therapy for cancer.

The Long Noncoding RNA DNM3OS Is a Reservoir of FibromiRs with Major Functions in Lung Fibroblast Response to TGF-ß and Pulmonary Fibrosis.

Rationale: Given the paucity of effective treatments for idiopathic pulmonary fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. TGF-ß (transforming growth factor-ß) is the main profibrotic factor, but its inhibition is associated with severe side effects because of its pleiotropic role. Objectives: To determine if downstream noncoding effectors of TGF-ß in fibroblasts may represent new effective therapeutic targets whose modulation may be well tolerated. Methods: We investigated the whole noncoding fraction of TGF-ß-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblasts. Differential expression of the long noncoding RNA (lncRNA) DNM3OS (dynamin 3 opposite strand) and its associated microRNAs (miRNAs) was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis. Measurements and Main Results: We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-ß-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e., miR-199a-5p/3p and miR-214-3p), which influence SMAD and non-SMAD components of TGF-ß signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis. Conclusions: Pharmacological approaches aiming at interfering with the lncRNA DNM3OS may represent new effective therapeutic strategies in IPF.

RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression.

Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

Detecting subtle transcriptomic perturbations induced by lncRNAs knock-down in single-cell CRISPRi screening using a new sparse supervised autoencoder neural network.

Single-cell CRISPR-based transcriptome screens are potent genetic tools for concomitantly assessing the expression profiles of cells targeted by a set of guides RNA (gRNA), and inferring target gene functions from the observed perturbations. However, due to various limitations, this approach lacks sensitivity in detecting weak perturbations and is essentially reliable when studying master regulators such as transcription factors. To overcome the challenge of detecting subtle gRNA induced transcriptomic perturbations and classifying the most responsive cells, we developed a new supervised autoencoder neural network method. Our Sparse supervised autoencoder (SSAE) neural network provides selection of both relevant features (genes) and actual perturbed cells. We applied this method on an in-house single-cell CRISPR-interference-based (CRISPRi) transcriptome screening (CROP-Seq) focusing on a subset of long non-coding RNAs (lncRNAs) regulated by hypoxia, a condition that promote tumor aggressiveness and drug resistance, in the context of lung adenocarcinoma (LUAD). The CROP-seq library of validated gRNA against a subset of lncRNAs and, as positive controls, HIF1A and HIF2A, the 2 main transcription factors of the hypoxic response, was transduced in A549 LUAD cells cultured in normoxia or exposed to hypoxic conditions during 3, 6 or 24 h. We first validated the SSAE approach on HIF1A and HIF2 by confirming the specific effect of their knock-down during the temporal switch of the hypoxic response. Next, the SSAE method was able to detect stable short hypoxia-dependent transcriptomic signatures induced by the knock-down of some lncRNAs candidates, outperforming previously published machine learning approaches. This proof of concept demonstrates the relevance of the SSAE approach for deciphering weak perturbations in single-cell transcriptomic data readout as part of CRISPR-based screening.

Pervasive role of the long noncoding RNA DNM3OS in development and diseases.

Thousands of unique noncoding RNAs (ncRNAs) are expressed in human cells, some are tissue or cell type specific whereas others are considered as house-keeping molecules. Studies over the last decade have modified our perception of ncRNAs from transcriptional noise to functional regulatory transcripts that influence a variety of molecular processes such as chromatin remodeling, transcription, post-transcriptional modifications, or signal transduction. Consequently, aberrant expression of many ncRNAs plays a causative role in the initiation and progression of various diseases. Since the identification of its developmental role, the long ncRNA DNM3OS (Dynamin 3 Opposite Strand) has attracted attention of researchers in distinct fields including oncology, fibroproliferative diseases, or bone disorders. Mechanistic studies have in particular revealed the multifaceted nature of DNM3OS and its important pathogenic role in several human disorders. In this review, we summarize the current knowledge of DNM3OS functions in diseases, with an emphasis on its potential as a novel therapeutic target. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

The immunogenicity of the tumor-associated antigen α-fetoprotein is enhanced by a fusion with a transmembrane domain.

AIM: To investigate the ability of recombinant modified vaccinia virus Ankara (rMVA) vector to induce an immune response against a well-tolerated self-antigen. METHODS: rMVA vectors expressing different form of α-fetoprotein (AFP) were produced and characterized. Naïve mice were vaccinated with MVA vectors expressing the AFP antigen in either a secreted, or a membrane-bound, or an intracellular form. The immune response was monitored by an IFNΓ ELISpot assay and antibody detection. RESULTS: Vaccination with the membrane-associated form of AFP induced a stronger CD8(+) T-cell response compared to the ones obtained with the MVA encoding the secreted or the intracellular forms of AFP. Moreover, the vaccination with the membrane-bound AFP elicited the production of AFP-specific antibodies. CONCLUSIONS: The AFP transmembrane form is more immunogenic. Expressing a membrane-bound form in the context of an MVA vaccination could enhance the immunogenicity of a self-antigen.

A role for metformin in the treatment of Dupuytren disease?

Dupuytren disease (DD) is a hand-localized fibrotic disorder characterized by a scar-like, collagen-rich cord. Treatment usually comprises surgical removal of the cord, but is associated with a high relapse rate, in some cases requiring finger amputation. There is currently no consensual medical approach for treating DD. Numerous preclinical studies have highlighted antifibrotic properties of metformin, and the aim of this study was to assess a potential antifibrotic role of metformin in DD. Fibroblasts from DD cords (DF) and phenotypically normal palmar fascia (PF) were extracted from surgical specimens and cultured. The fibrotic status of DF and PF was compared at baseline, and under profibrotic (TGF-ß stimulation) and antifibrotic (metformin stimulation) conditions, using quantitative RT-PCR, western blot, immunocytochemistry, and a functional fibroblast contraction assay. At baseline, DF showed higher levels of fibrotic markers and contraction capacity compared with PF. Both types of fibroblasts responded to TGF-ß stimulation. Treatment of DF and PF with metformin did not affect basal levels of fibrotic markers and contraction but largely prevented their induction by TGF-ß. In conclusion, our data show that metformin inhibits TGF-ß-induced expression of fibrotic markers and contraction in hand-derived fibroblasts. This supports the case for a clinical trial to assess the repurposing of metformin as an adjuvant to surgery, to prevent, reduce, or delay recurrence in at-risk DD patients.

Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma.

Lineage dedifferentiation toward a mesenchymal-like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and acquired resistance to targeted therapy in melanoma. Here, we show that the anti-fibrotic drug nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy, and delay tumor relapse in a preclinical model of melanoma. Acquisition of this resistant phenotype and its reversion by nintedanib pointed to miR-143/-145 pro-fibrotic cluster as a driver of this mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p, collaborated to mediate transition toward a drug-resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof of principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy.

AFP-specific immunotherapy impairs growth of autochthonous hepatocellular carcinoma in mice.

BACKGROUND AND AIMS: In this study, we have assessed the potential of antigen-specific immunotherapy against hepatocellular carcinoma (HCC) in conditions of low tumour burden, in an autochthonous HCC model. METHODS: Diethylnitrosamine (DEN) injected into infant mice results in the development of multi-nodular HCC in which alpha-fetoprotein (AFP) is re-expressed. DEN-injected animals received an antigen-specific immunization with a synthetic vector consisting of a low dose of AFP-encoding plasmid formulated with the amphiphilic block copolymer 704 (DNAmAFP/704). Animals were treated at 4 and 5 months, before macroscopic nodules were detected, and were sacrificed at 8 months. The tumour burden, as well as liver histology, was assessed. AFP and MHC class I molecule expression in the nodules were monitored by qRT-PCR. RESULTS: The AFP-specific immunotherapy led to a significant (65%) reduction in tumour size. The reduced expression of AFP and MHC class I molecules was measured in the remaining nodules taken from the DNAmAFP/704-treated group. CONCLUSIONS: This is the first study demonstrating the relevance of antigen-specific immunotherapy in an autochthonous HCC model. In this context, we validated the use of an anti-tumour immunotherapy based on vaccination with nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer. Our results demonstrate the potential of this strategy as adjuvant immunotherapy to reduce the recurrence risk after local treatment of HCC patients.

Differential biodistribution of oncolytic poxvirus administered systemically in an autochthonous model of hepatocellular carcinoma.

BACKGROUND: Preclinical studies have demonstrated that, unlike oncolytic adenoviruses, oncolytic vaccinia viruses can reach implanted tumors upon systemic injection. However, the biodistribution of this oncolytic agent in in situ autochthonous tumor models remains poorly characterized. In the present study, we assessed this biodistribution in a model of mouse hepatocellular carcinoma (HCC) obtained after injection of the carcinogen diethylnitrosamine (DEN). METHODS: Twelve months after DEN administration, histology, quantitative reverse transcription-polymerase chain reaction, in situ hybridization and viral titration were used to characterize tumors, as well as to assess the viral load of the livers upon either intravenous or intraperitoineal injection. RESULTS: The results obtained showed that the architecture of the liver was lost, with a noticeable absence of sinusoids, as well as the presence of steatosis and α-fetoprotein-positive HCC tumor nodules. Bioluminescence imaging and measures of the infective virus load demonstrated that intravenous injection of 10(8) plaque-forming units of the recombinant vaccinia virus led to a predominant transduction of the liver, whereas intraperitoneal injection resulted in a lower level of liver transduction accompanied by an increased infection of the lungs, spleen, kidneys and bowels. Immunohistochemical analysis of liver sections of animals injected intravenously with the virus revealed a preferential localization of vaccinia-specific immunoreactivity in the tumors. CONCLUSIONS: The findings of the present study emphasize the importance of the route of administration of the vector and highlight the relevance of systemic injection of oncolytic vaccinia virus in the context of hepatocellular carcinoma.

Therapeutic effect of sodium iodide symporter gene therapy combined with external beam radiotherapy and targeted drugs that inhibit DNA repair.

Adenoviral (AdV) transfer of sodium iodide symporter (NIS) gene has translational potential, but relatively low levels of transduction and subsequent radioisotope uptake limit the efficacy of the approach. In previous studies, we showed that combining NIS gene delivery with external beam radiotherapy (EBRT) and DNA damage repair inhibitors increased viral gene expression and radioiodide uptake. Here, we report the therapeutic efficacy of this strategy. An adenovirus expressing NIS from a telomerase promoter (Ad-hTR-NIS) was cytotoxic combined with relatively high-dose (50 microCi) (131)I therapy and enhanced the efficacy of EBRT combined with low-dose (10 and 25 microCi) (131)I therapy in colorectal and head and neck cancer cells. Combining this approach with ataxia-telangiectasia mutated (ATM) or DNA-dependent protein kinase (DNA-PK) inhibition caused maintenance of double-stranded DNA breaks (DSBs) at 24 hours and increased cytotoxicity on clonogenic assay. When the triplet of NIS-mediated (131)I therapy, EBRT, and DNA-PKi was used in vivo, 90% of mice were tumor-free at 5 weeks. Acute radiation toxicity in the EBRT field was not exacerbated. In contrast, DNA-PKi did not enhance the therapeutic efficacy of EBRT plus adenovirus-mediated HSVtk/ganciclovir (GCV). Therefore, combining NIS gene therapy and EBRT represents an ideal strategy to exploit the therapeutic benefits of novel radiosensitizers.

The FibromiR miR-214-3p Is Upregulated in Duchenne Muscular Dystrophy and Promotes Differentiation of Human Fibro-Adipogenic Muscle Progenitors.

Fibrosis is a deleterious invasion of tissues associated with many pathological conditions, such as Duchenne muscular dystrophy (DMD) for which no cure is at present available for its prevention or its treatment. Fibro-adipogenic progenitors (FAPs) are resident cells in the human skeletal muscle and can differentiate into myofibroblasts, which represent the key cell population responsible for fibrosis. In this study, we delineated the pool of microRNAs (miRNAs) that are specifically modulated by TGFß1 in FAPs versus myogenic progenitors (MPs) by a global miRNome analysis. A subset of candidates, including several "FibromiRs", was found differentially expressed between FAPs and MPs and was also deregulated in DMD versus healthy biopsies. Among them, the expression of the TGFß1-induced miR-199a~214 cluster was strongly correlated with the fibrotic score in DMD biopsies. Loss-of-function experiments in FAPs indicated that a miR-214-3p inhibitor efficiently blocked expression of fibrogenic markers in both basal conditions and following TGFß1 stimulation. We found that FGFR1 is a functional target of miR-214-3p, preventing the signaling of the anti-fibrotic FGF2 pathway during FAP fibrogenesis. Overall, our work demonstrates that the « FibromiR ¼ miR-214-3p is a key activator of FAP fibrogenesis by modulating the FGF2/FGFR1/TGFß axis, opening new avenues for the treatment of DMD.

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples.

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

Correction: The nuclear hypoxia-regulated NLUCAT1 long non-coding RNA contributes to an aggressive phenotype in lung adenocarcinoma through regulation of oxidative stress.

Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. Hypoxic regions within tumors represent sources of aggressiveness and resistance to therapy. Although long non-coding RNAs (lncRNAs) are increasingly recognized as major gene expression regulators, their regulation and function following hypoxic stress are still largely unexplored. Combining profiling studies on early-stage lung adenocarcinoma (LUAD) biopsies and on A549 LUAD cell lines cultured in normoxic or hypoxic conditions, we identified a subset of lncRNAs that are both correlated with the hypoxic status of tumors and regulated by hypoxia in vitro. We focused on a new transcript, Nuclear LUCAT1 (NLUCAT1), which is strongly upregulated by hypoxia in vitro and correlated with hypoxic markers and poor prognosis in LUADs. Full molecular characterization showed that NLUCAT1 is a large nuclear transcript composed of six exons and mainly regulated by NF-κB and NRF2 transcription factors. CRISPR-Cas9-mediated invalidation of NLUCAT1 revealed a decrease in proliferative and invasive properties, an increase in oxidative stress and a higher sensitivity to cisplatin-induced apoptosis. Transcriptome analysis of NLUCAT1-deficient cells showed repressed genes within the antioxidant and/or cisplatin-response networks. We demonstrated that the concomitant knockdown of four of these genes products, GPX2, GLRX, ALDH3A1, and PDK4, significantly increased ROS-dependent caspase activation, thus partially mimicking the consequences of NLUCAT1 inactivation in LUAD cells. Overall, we demonstrate that NLUCAT1 contributes to an aggressive phenotype in early-stage hypoxic tumors, suggesting it may represent a new potential therapeutic target in LUADs.

In vivo retargeting of adenovirus type 5 to alphavbeta6 integrin results in reduced hepatotoxicity and improved tumor uptake following systemic delivery.

A key impediment to successful cancer therapy with adenoviral vectors is the inefficient transduction of malignant tissue in vivo. Compounding this problem is the lack of cancer-specific targets, coupled with a shortage of corresponding high-efficiency ligands, permitting selective retargeting. The epithelial cell-specific integrin alphavbeta6 represents an attractive target for directed therapy since it is generally not expressed on normal epithelium but is upregulated in numerous carcinomas, where it plays a role in tumor progression. We previously have characterized a high-affinity, alphavbeta6-selective peptide (A20FMDV2) derived from VP1 of foot-and-mouth disease virus. We generated recombinant adenovirus type 5 (Ad5) fiber knob, incorporating A20FMDV2 in the HI loop, for which we validated the selectivity of binding and functional inhibition of alphavbeta6. The corresponding alphavbeta6-retargeted virus Ad5-EGFP(A20) exhibited up to 50-fold increases in coxsackievirus- and-adenovirus-receptor-independent transduction and up to 480-fold-increased cytotoxicity on a panel of alphavbeta6-positive human carcinoma lines compared with Ad5-EGFP(WT). Using an alphavbeta6-positive (DX3-beta6) xenograft model, we observed a approximately 2-fold enhancement in tumor uptake over Ad5-EGFP(WT) following systemic delivery. Furthermore, approximately 5-fold-fewer Ad5-EGFP(A20) genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. Warfarin pretreatment, to deplete coagulation factors, did not improve tumor uptake significantly with either virus but did significantly reduce liver sequestration and hepatic toxicity. The ability of Ad5-EGFP(A20) to improve delivery to alphavbeta6, combined with its reduced hepatic tropism and toxicity, highlights its potential as a prototype virus for future clinical investigation.

Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours.

PURPOSE: In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. METHODS: We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. RESULTS: In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. CONCLUSION: This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects.

Proteomic Analysis of Iodinated Contrast Agent-Induced Perturbation of Thyroid Iodide Uptake.

(1) Background: We recently showed that iodinated contrast media (ICM) reduced thyroid uptake of iodide independently of free iodide through a mechanism different from that of NaI and involving a dramatic and long-lasting decrease in Na/I symporter expression. The present study aimed at comparing the response of the thyroid to ICM and NaI using a quantitative proteomic approach. (2) Methods: Scintiscans were performed on ICM-treated patients. Micro Single-Photon Emission Computed Tomography (microSPECT/CT) imaging was used to assess thyroid uptakes in ICM- or NaI-treated mice and their response to recombinant human thyroid-stimulating hormone. Total thyroid iodide content and proteome was determined in control, NaI-, or ICM-treated animals. (3) Results: The inhibitory effect of ICM in patients was selectively observed on thyroids but not on salivary glands for up to two months after a systemic administration. An elevated level of iodide was observed in thyroids from NaI-treated mice but not in those from ICM animals. Exposure of the thyroid to NaI modulates 15 cellular pathways, most of which are also affected by ICM treatment (including the elF4 and P706SK cell signaling pathway and INSR identified as an upstream activator in both treatments). In addition, ICM modulates 16 distinct pathways and failed to affect thyroid iodide content. Finally, administration of ICM reduces thyroid-stimulating hormone (TSH) receptor expression which results in a loss of TSH-induced iodide uptake by the thyroid. (4) Conclusions: Common intracellular mechanisms are involved in the ICM- and NaI-induced reduction of iodide uptake. However, ICM fails to affect thyroid iodide content which suggests that the modulation of these common pathways is triggered by separate effectors. ICM also modulates numerous distinct pathways which may account for its long-lasting effect on thyroid uptake. These observations may have implications in the management of patients affected by differentiated thyroid carcinomas who have been exposed to ICM. They also provide the basis for the utilization of ICM-based compounds in radioprotection of the thyroid.

Therapeutic potential of replication-selective oncolytic adenoviruses on cells from familial and sporadic desmoid tumors.

PURPOSE: Constitutive activation of the Wnt signaling pathway is a hallmark of many cancers and has been associated with familial and sporadic desmoid tumors. The aim of the present study is to assess the therapeutic potential of oncolytic adenoviruses selectively replicating in cells in which the Wnt signaling pathway is active on primary cells from desmoid tumors. EXPERIMENTAL DESIGN: Primary cells extracted from familial (n = 3) or sporadic (n = 3) desmoid tumors were cultured short term. Cancer cell survival and viral replication were measured in vitro upon infection with two different oncolytic adenoviruses targeting a constitutive activation of the Wnt signaling pathway. Adenoviral infectivity was also assessed. RESULTS: Although cells extracted from one sporadic desmoid tumor responded very well to the oncolytic action of the adenoviruses (<20% of viable cells upon infection at a multiplicity of infection of 10), cells from two tumor samples were totally resistant to the viral action. Cells from the remaining samples showed intermediate sensitivity to the oncolytic viruses. These effects were correlated to the level of infectivity of the cells. Finally, in responder cells, evidences of viral replication was observed. CONCLUSIONS: Our experimental data suggest that the response of desmoid tumor cells to oncolytic adenovirus is neither correlated to the type of mutation activating the Wnt signaling pathway nor to the familial or sporadic nature of the tumor. In addition, they highlight the variability of infectivity of individual tumors and predict a great variability in the response to oncolytic adenoviruses.