Type 1 interferons and Foxo1 down-regulation play a key role in age-related T-cell exhaustion in mice.

Foxo family transcription factors are critically involved in multiple processes, such as metabolism, quiescence, cell survival and cell differentiation. Although continuous, high activity of Foxo transcription factors extends the life span of some species, the involvement of Foxo proteins in mammalian aging remains to be determined. Here, we show that Foxo1 is down-regulated with age in mouse T cells. This down-regulation of Foxo1 in T cells may contribute to the disruption of naive T-cell homeostasis with age, leading to an increase in the number of memory T cells. Foxo1 down-regulation is also associated with the up-regulation of co-inhibitory receptors by memory T cells and exhaustion in aged mice. Using adoptive transfer experiments, we show that the age-dependent down-regulation of Foxo1 in T cells is mediated by T-cell-extrinsic cues, including type 1 interferons. Taken together, our data suggest that type 1 interferon-induced Foxo1 down-regulation is likely to contribute significantly to T-cell dysfunction in aged mice.

Alteration of the embryonic microenvironment and sex-specific responses of the preimplantation embryo related to a maternal high-fat diet in the rabbit model.

The maternal metabolic environment can be detrimental to the health of the offspring. In a previous work, we showed that maternal high-fat (HH) feeding in rabbit induced sex-dependent metabolic adaptation in the fetus and led to metabolic syndrome in adult offspring. As early development representing a critical window of susceptibility, in the present work we aimed to explore the effects of the HH diet on the oocyte, preimplantation embryo and its microenvironment. In oocytes from females on HH diet, transcriptomic analysis revealed a weak modification in the content of transcripts mainly involved in meiosis and translational control. The effect of maternal HH diet on the embryonic microenvironment was investigated by identifying the metabolite composition of uterine and embryonic fluids collected in vivo by biomicroscopy. Metabolomic analysis revealed differences in the HH uterine fluid surrounding the embryo, with increased pyruvate concentration. Within the blastocoelic fluid, metabolomic profiles showed decreased glucose and alanine concentrations. In addition, the blastocyst transcriptome showed under-expression of genes and pathways involved in lipid, glucose and amino acid transport and metabolism, most pronounced in female embryos. This work demonstrates that the maternal HH diet disrupts the in vivo composition of the embryonic microenvironment, where the presence of nutrients is increased. In contrast to this nutrient-rich environment, the embryo presents a decrease in nutrient sensing and metabolism suggesting a potential protective process. In addition, this work identifies a very early sex-specific response to the maternal HH diet, from the blastocyst stage.

Sunitinib-induced cardiac hypertrophy and the endothelin axis.

Anti-angiogenics drugs in clinical use for cancer treatment induce cardiotoxic side effects. The endothelin axis is involved in hypertension and cardiac remodelling, and addition of an endothelin receptor antagonist to the anti-angiogenic sunitinib was shown to reduce cardiotoxicity of sunitinib in mice. Here, we explored further the antidote effect of the endothelin receptor antagonist macitentan in sunitinib-treated animals on cardiac remodeling. Methods: Tumor-bearing mice treated per os daily by sunitinib or vehicle were imaged before and after 1, 3 and 6 weeks of treatment by positron emission tomography using [18F]fluorodeoxyglucose and by echocardiography. Non-tumor-bearing animals were randomly assigned to be treated per os daily by vehicle or sunitinib or macitentan or sunitinib+macitentan, and imaged by echocardiography after 5 weeks. Hearts were harvested for histology and molecular analysis at the end of in vivo exploration. Results: Sunitinib treatment increases left ventricular mass and ejection fraction and induces cardiac fibrosis. Sunitinib also induces an early increase in cardiac uptake of [18F]fluorodeoxyglucose, which is significantly correlated with increased left ventricular mass at the end of treatment. Co-administration of macitentan prevents sunitinib-induced hypertension, increase in ejection fraction and cardiac fibrosis, but fails to prevent increase of the left ventricular mass. Conclusion: Early metabolic changes predict sunitinib-induced cardiac remodeling. Endothelin blockade can prevent some but not all cardiotoxic side-effects of sunitinib, in particular left ventricle hypertrophy that appears to be induced by sunitinib through an endothelin-independent mechanism.

Metabolomic differences in blastocoel and uterine fluids collected in vivo by ultrasound biomicroscopy on rabbit embryos†.

The success of embryo development and implantation depends in part on the environment in which the embryo evolves. However, the composition of the uterine fluid surrounding the embryo in the peri-implantation period remains poorly studied. In this work, we aimed to develop a new strategy to visualize, collect, and analyze both blastocoelic liquid and juxta-embryonic uterine fluid from in vivo peri-implantation rabbit embryos. Using high-resolution ultrasound biomicroscopy, embryos were observed as fluid-filled anechoic vesicles, some of which were surrounded by a thin layer of uterine fluid. Ultrasound-guided puncture and aspiration of both the blastocoelic fluid contained in the embryo and the uterine fluid in the vicinity of the embryo were performed. Using nuclear magnetic resonance spectroscopy, altogether 24 metabolites were identified and quantified, of which 21 were detected in both fluids with a higher concentration in the uterus compared to the blastocoel. In contrast, pyruvate was detected at a higher concentration in blastocoelic compared to uterine fluid. Two acidic amino acids, glutamate and aspartate, were not detected in uterine fluid in contrast to blastocoelic fluid, suggesting a local regulation of uterine fluid composition. To our knowledge, this is the first report of simultaneous analysis of blastocoelic and uterine fluids collected in vivo at the time of implantation in mammals, shedding new insight for understanding the relationship between the embryo and its local environment at this critical period of development.

Identification of a New QTL Region on Mouse Chromosome 1 Responsible for Male Hypofertility: Phenotype Characterization and Candidate Genes.

Male fertility disorders often have their origin in disturbed spermatogenesis, which can be induced by genetic factors. In this study, we used interspecific recombinant congenic mouse strains (IRCS) to identify genes responsible for male infertility. Using ultrasonography, in vivo and in vitro fertilization (IVF) and electron microscopy, the phenotyping of several IRCS carrying mouse chromosome 1 segments of Mus spretus origin revealed a decrease in the ability of sperm to fertilize. This teratozoospermia included the abnormal anchoring of the acrosome to the nucleus and a persistence of residual bodies at the level of epididymal sperm midpiece. We identified a quantitative trait locus (QTL) responsible for these phenotypes and we have proposed a short list of candidate genes specifically expressed in spermatids. The future functional validation of candidate genes should allow the identification of new genes and mechanisms involved in male infertility.

Dullard-mediated Smad1/5/8 inhibition controls mouse cardiac neural crest cells condensation and outflow tract septation.

The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1. Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot.

Long-term cardiovascular disorders in the STOX1 mouse model of preeclampsia.

Adverse long-term cardiovascular (CV) consequences of PE are well established in women. However, the mechanism responsible for that risk remains unknown. Here, we mated wild-type female mice of the FVB/N strain to STOX1A-overexpressing mice to mimic severe PE and investigated the long-term consequences on the maternal cardiovascular system. Ultrasonography parameters were analyzed in mice before pregnancy and at 3 and 6 months post-pregnancy. At 6 months post-pregnancy, cardiac stress test induced by dobutamine injection revealed an abnormal ultrasonography Doppler profile in mice with previous PE. Eight months post-pregnancy, the heart, endothelial cells (ECs) and plasma of females were analyzed and compared to controls. The heart of mice with PE showed left-ventricular hypertrophy associated with altered histology (fibrosis). Transcriptomic analysis revealed the deregulation of 1149 genes in purified ECs and of 165 genes in the hearts, many being involved in heart hypertrophy. In ECs, the upregulated genes were associated with inflammation and cellular stress. Systems biology analysis identified interleukin 6 (IL-6) as a hub gene connecting these pathways. Plasma profiling of 33 cytokines showed that, 8 of them (Cxcl13, Cxcl16, Cxcl11, IL-16, IL-10, IL-2, IL-4 and Ccl1) allowed to discriminate mice with previous PE from controls. Thus, PE triggers female long-term CV consequences on the STOX1 mouse model.

Standardised imaging pipeline for phenotyping mouse laterality defects and associated heart malformations, at multiple scales and multiple stages.

Laterality defects are developmental disorders resulting from aberrant left/right patterning. In the most severe cases, such as in heterotaxy, they are associated with complex malformations of the heart. Advances in understanding the underlying physiopathological mechanisms have been hindered by the lack of a standardised and exhaustive procedure in mouse models for phenotyping left/right asymmetries of all visceral organs. Here, we have developed a multimodality imaging pipeline, which combines non-invasive micro-ultrasound imaging, micro-computed tomography (micro-CT) and high-resolution episcopic microscopy (HREM) to acquire 3D images at multiple stages of development and at multiple scales. On the basis of the position in the uterine horns, we track in a single individual, the progression of organ asymmetry, the situs of all visceral organs in the thoracic or abdominal environment, and the fine anatomical left/right asymmetries of cardiac segments. We provide reference anatomical images and organ reconstructions in the mouse, and discuss differences with humans. This standardised pipeline, which we validated in a mouse model of heterotaxy, offers a fast and easy-to-implement framework. The extensive 3D phenotyping of organ asymmetry in the mouse uses the clinical nomenclature for direct comparison with patient phenotypes. It is compatible with automated and quantitative image analyses, which is essential to compare mutant phenotypes with incomplete penetrance and to gain mechanistic insight into laterality defects.

Preeclampsia induced by STOX1 overexpression in mice induces intrauterine growth restriction, abnormal ultrasonography and BOLD MRI signatures.

BACKGROUND: Preeclampsia is a major hypertensive disease caused by pregnancy, inducing proteinuria and increased blood pressure starting from the second half of pregnancy (early preeclampsia) or near the end of pregnancy (late preeclampsia). Pre-symptomatic diagnosis would allow for therapeutic interventions, such as with low-dose aspirin. Among non-invasive methods to explore organ physiology, Doppler ultrasonography (US) and functional blood oxygenation level-dependent (BOLD) MRI (which do not need radioactive contrast agents such as gadolinium) can be used in pregnant women. METHODS: In this study, we used US and BOLD MRI to finely characterize the phenotype of preeclampsia induced by the foeto-placental overexpression of the transcription factor storkhead box 1A (STOX1A) in female mice. RESULTS: We could observe late fetal growth restriction consistent with the placental dysfunction revealed by US and the known association between preeclampsia and intra-uterine growth restriction. On US, uterine and umbilical artery as well as heart and kidney parameters were modified in preeclamptic mice. On BOLD MRI, mean T2* values revealed considerable differences between control and preeclamptic placentas, which suggests altered dynamics of oxygen release and ratio of oxyhemoglobin to deoxyhemoglobin in the model. CONCLUSION: These preliminary pre-clinical results suggest that BOLD MRI could be evaluated as a prognostic/diagnostic tool for preeclampsia.

Wnt proteins contribute to neuromuscular junction formation through distinct signaling pathways.

Understanding the developmental steps that shape formation of the neuromuscular junction (NMJ) connecting motoneurons to skeletal muscle fibers is crucial. Wnt morphogens are key players in the formation of this specialized peripheral synapse, but their individual and collaborative functions and downstream pathways remain poorly understood at the NMJ. Here, we demonstrate through Wnt4 and Wnt11 gain-of-function studies in cell culture or in mice that Wnts enhance acetylcholine receptor (AChR) clustering and motor axon outgrowth. By contrast, loss of Wnt11 or Wnt-dependent signaling in vivo decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR mRNA levels and AChR clustering downstream of activation of the ß-catenin pathway. Strikingly, Wnt4 and Wnt11 co-immunoprecipitate with Vangl2, a core component of the planar cell polarity (PCP) pathway, which accumulates at embryonic NMJs. Moreover, mice bearing a Vangl2 loss-of-function mutation (loop-tail) exhibit fewer AChR clusters and overgrowth of motor axons bypassing AChR clusters. Together, our results provide genetic and biochemical evidence that Wnt4 and Wnt11 cooperatively contribute to mammalian NMJ formation through activation of both the canonical and Vangl2-dependent core PCP pathways.

Tumor Stiffening, a Key Determinant of Tumor Progression, is Reversed by Nanomaterial-Induced Photothermal Therapy.

Tumor stiffening, stemming from aberrant production and organization of extracellular matrix (ECM), has been considered a predictive marker of tumor malignancy, non-invasively assessed by ultrasound shear wave elastography (SWE). Being more than a passive marker, tumor stiffening restricts the delivery of diagnostic and therapeutic agents to the tumor and per se could modulate cellular mechano-signaling, tissue inflammation and tumor progression. Current strategies to modify the tumor extracellular matrix are based on ECM-targeting chemical agents but also showed deleterious systemic effects. On-demand excitable nanomaterials have shown their ability to perturb the tumor microenvironment in a spatiotemporal-controlled manner and synergistically with chemotherapy. Here, we investigated the evolution of tumor stiffness as well as tumor integrity and progression, under the effect of mild hyperthermia and thermal ablation generated by light-exposed multi-walled carbon nanotubes (MWCNTs) in an epidermoid carcinoma mouse xenograft. SWE was used for real-time mapping of the tumor stiffness, both during the two near infrared irradiation sessions and over the days after the treatment. We observed a transient and reversible stiffening of the tumor tissue during laser irradiation, which was lowered at the second session of mild hyperthermia or photoablation. In contrast, over the days following photothermal treatment, the treated tumors exhibited a significant softening together with volume reduction, whereas non-treated growing tumors showed an increase of tumor rigidity. The organization of the collagen matrix and the distribution of CNTs revealed a spatio-temporal correlation between the presence of nanoheaters and the damages on collagen and cells. This study highlights nanohyperthermia as a promising adjuvant strategy to reverse tumor stiffening and normalize the mechanical tumor environment.

Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion.

Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF.

Antitumour activity of an inhibitor of miR-34a in liver cancer with ß-catenin-mutations.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the most prevalent primary tumour of the liver. About a third of these tumours presents activating mutations of the ß-catenin gene. The molecular pathogenesis of HCC has been elucidated, but mortality remains high, and new therapeutic approaches, including treatments based on microRNAs, are required. We aimed to identify candidate microRNAs, regulated by ß-catenin, potentially involved in liver tumorigenesis. DESIGN: We used a mouse model, in which ß-catenin signalling was overactivated exclusively in the liver by the tamoxifen-inducible and Cre-Lox-mediated inactivation of the Apc gene. This model develops tumours with properties similar to human HCC. RESULTS: We found that miR-34a was regulated by ß-catenin, and significantly induced by the overactivation of ß-catenin signalling in mouse tumours and in patients with HCC. An inhibitor of miR-34a (locked nucleic acid, LNA-34a) exerted antiproliferative activity in primary cultures of hepatocyte. This inhibition of proliferation was associated with a decrease in cyclin D1 levels, orchestrated principally by HNF-4α, a target of miR-34a considered to act as a tumour suppressor in the liver. In vivo, LNA-34a approximately halved progression rates for tumours displaying ß-catenin activation together with an activation of caspases 2 and 3. CONCLUSIONS: This work demonstrates the key oncogenic role of miR-34a in liver tumours with ß-catenin gene mutations. We suggest that patients diagnosed with HCC with ß-catenin mutations could be treated with an inhibitor of miR-34a. The potential value of this strategy lies in the modulation of the tumour suppressor HNF-4α, which targets cyclin D1, and the induction of a proapoptotic programme.

Resveratrol Decreases TXNIP mRNA and Protein Nuclear Expressions With an Arterial Function Improvement in Old Mice.

Aging leads to a high prevalence of glucose intolerance and cardiovascular diseases, with oxidative stress playing a potential role. Resveratrol has shown promising effects on glucose tolerance and tends to improve endothelial function in elderly patients. Thioredoxin-interacting protein (TXNIP) was recently proposed as a potential link connecting glucose metabolism to oxidative stress. Here, we investigated the resveratrol-induced improvement of arterial aging phenotype in old mice and the expression of aortic TXNIP. Using an in vivo model of old mice with or without 3-month resveratrol treatment, we investigated the effects of resveratrol on age-related impairments from a cardiovascular Doppler analysis, to a molecular level, by studying inflammation and oxidative stress factors. We found a dual effect of resveratrol, with a decrease of age-related glucose intolerance and oxidative stress imbalance leading to reduced matrix remodeling that forestalls arterial aging phenotype in terms of intima-media thickness and arterial distensibility. These results provide the first evidence that aortic TXNIP mRNA and protein nuclear expressions are increased in the arterial aging and decreased by resveratrol treatment. In conclusion, we demonstrated that resveratrol helped to restore several aging impaired processes in old mice, with a decrease of aortic TXNIP mRNA and protein nuclear expressions.

Intestinal inhibition of Atg7 prevents tumour initiation through a microbiome-influenced immune response and suppresses tumour growth.

Here, we show that autophagy is activated in the intestinal epithelium in murine and human colorectal cancer and that the conditional inactivation of Atg7 in intestinal epithelial cells inhibits the formation of pre-cancerous lesions in Apc(+/-) mice by enhancing anti-tumour responses. The antibody-mediated depletion of CD8(+) T cells showed that these cells are essential for the anti-tumoral responses mediated by the inhibition of autophagy. We show that Atg7 deficiency leads to intestinal dysbiosis and that the microbiota is required for anticancer responses. In addition, Atg7 deficiency resulted in a stress response accompanied by metabolic defects, AMPK activation and p53-mediated cell-cycle arrest in tumour cells but not in normal tissue. This study reveals that the inhibition of autophagy within the epithelium may prevent the development and progression of colorectal cancer in genetically predisposed patients.

Targeting CD226/DNAX accessory molecule-1 (DNAM-1) in collagen-induced arthritis mouse models.

BACKGROUND: Genetic studies have pointed out that CD226 variants, encoding DNAM-1, could be associated with susceptibility to rheumatoid arthritis. Therefore, we aimed to determine the influence of DNAM-1 on the development of arthritis using the collagen-induced arthritis (CIA) mouse model. METHODS: CIA was induced in mice on a DBA/1 background, treated in parallel with a DNAM-1 neutralizing monoclonal antibody, a control IgG and PBS, respectively. CIA was also induced in mice deficient for DNAM-1(dnam1-/-) and control dnam-1+/+ mice on a C57/BL6 background. Mice were monitored for clinical and ultrasound signs of arthritis. Histological analysis was performed to search for inflammatory infiltrates and erosions. The Mann-Whitney U test for non-related samples was used for statistical analysis. RESULTS: There was a non-significant trend for a less arthritic phenotype in mice receiving anti-DNAM-1 mAb at both clinical, ultrasound and histological assessments. But, we did not observe any difference between dnam1+/+ and dnam1-/- mice for incidence nor severity of clinical arthritis. Histological analysis revealed inflammatory scores similar in both groups, without evidence of erosion. Collagen antibodies levels were similar in all mice, confirming immunization with collagen. CONCLUSION: Despite some clues suggesting a role of DNAM-1 in arthritis, these complementary approaches demonstrate no contribution of CD226/DNAM-1 in the arthritic phenotype. These results contrast with previous studies showing a role in vivo of DNAM-1 in some autoimmune disorders.

Performance of skin ultrasound to measure skin involvement in different animal models of systemic sclerosis.

Animal models are widely used in systemic sclerosis (SSc) research. We set out to determine whether ultrasonography (US) could be used to assess skin fibrosis in two complementary SSc-models: the bleomycin-induced dermal fibrosis model and the tight-skin 1 mouse model. Back skin thickness was measured using a high-frequency ultrasound dedicated to the small animal. There was no significant difference in dermal thickness measured by US between mice injected with bleomycin and those treated with NaCl. These results were inconsistent with histological analyses. Mean US hypodermal thickness was significantly higher in tight-skin 1 mice as compared with Pa/Pa control subgroup (p = 0.02). Histologic and US measures of dermal and hypodermal thicknesses in this model were well correlated (r = 0.79). The intra-observer concordance was 0.96 for hypodermal thickness. US is reliable and sensitive in detecting hypodermal thickening in the tight-skin 1 mouse model. Further larger studies are warranted to better determine the place of US in SSc-research.

The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction.

OBJECTIVE: Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and overwhelming infiltration of innate immune cells has been shown to promote adverse remodeling and cardiac rupture. Recruitment of inflammatory cells in the ischemic heart depends highly on the family of CC-chemokines and their receptors. Here, we hypothesized that the chemokine decoy receptor D6, which specifically binds and scavenges inflammatory CC-chemokines, might limit inflammation and adverse cardiac remodeling after infarction. METHODS AND RESULTS: D6 was expressed in human and murine infarcted myocardium. In a murine model of myocardial infarction, D6 deficiency led to increased chemokine (C-C motif) ligand 2 and chemokine (C-C motif) ligand 3 levels in the ischemic heart. D6-deficient (D6(-/-)) infarcts displayed increased infiltration of pathogenic neutrophils and Ly6Chi monocytes, associated with strong matrix metalloproteinase-9 and matrix metalloproteinase-2 activities in the ischemic heart. D6(-/-) mice were cardiac rupture prone after myocardial infarction, and functional analysis revealed that D6(-/-) hearts had features of adverse remodeling with left ventricle dilation and reduced ejection fraction. Bone marrow chimera experiments showed that leukocyte-borne D6 had no role in this setting, and that leukocyte-specific chemokine (C-C motif) receptor 2 deficiency rescued the adverse phenotype observed in D6(-/-) mice. CONCLUSIONS: We show for the first time that the chemokine decoy receptor D6 limits CC-chemokine-dependent pathogenic inflammation and is required for adequate cardiac remodeling after myocardial infarction.

Delivery of mengovirus-derived RNA replicons into tumoural liver enhances the anti-tumour efficacy of a peripheral peptide-based vaccine.

Hepatocellular carcinoma is a deadly cancer with growing incidence for which immunotherapy is one of the most promising therapeutic approach. Peptide-based vaccines designed to induce strong, sustained CD8+ T cell responses are effective in animal models and cancer patients. We demonstrated the efficacy of curative peptide-based immunisation against a unique epitope of SV40 tumour antigen, through the induction of a strong CD8+ T cell-specific response, in our liver tumour model. However, as in human clinical trials, most tumour antigen epitopes did not induce a therapeutic effect, despite inducing strong CD8+ T cell responses. We therefore modified the tumour environment to enhance peptide-based vaccine efficacy by delivering mengovirus (MV)-derived RNA autoreplicating sequences (MV-RNA replicons) into the liver. The injection of replication-competent RNA replicons into the liver converted partial tumour regression into tumour eradication, whereas non-replicating RNA had no such effect. Replicating RNA replicon injection induced local recruitment of innate immunity effectors (NK and NKT) to the tumour and did not affect specific CD8+ T cell populations or other myelolymphoid subsets. The local delivery of such RNA replicons into tumour stroma is therefore a promising strategy complementary to the use of peripheral peptide-based vaccines for treating liver tumours.

Regression of established liver tumor induced by monoepitopic peptide-based immunotherapy.

Most types of cancer are difficult to eradicate, and some, like hepatocellular carcinoma, are almost always fatal. Among various interventions to improve the survival of patients with cancer, immunotherapy seems to hold some promises. However, it requires relevant animal models for preclinical development. In this study we report a new and relevant experimental model where liver tumors grow inside a nontumoral parenchyma of adult mice. This model is based on the intrasplenic injection in syngeneic recipient mice of hepatocytes from transgenic mice expressing SV40 large T oncogene specifically in the liver. Using this model where no apparent spontaneous cellular immune response was observed, immunization using a single injection of monoepitopic SV40 T Ag short peptide was sufficient to provoke liver tumor destruction, leading rapidly to complete remission. Tumor regression was associated with the induction of a long-lasting CD8+ T cell response, observed not only in the spleen but also, more importantly, in the tumoral liver. These results show the efficacy of peptide immunotherapy in the treatment of liver cancer.