Long-term quality of life is better after laparoscopic compared to open pancreatoduodenectomy.

BACKGROUND: Three randomized controlled trials have reported improved functional recovery after Laparoscopic pancreatoduodenectomy (LPD), as compared to open pancreatoduodenectomy (OPD). Long-term results regarding quality of life (QoL) are lacking. The aim of this study was to compare long-term QoL of LPD versus OPD. METHODS AND PATIENTS: A monocentric retrospective cross-sectional study was performed among patients < 75 years old who underwent LPD or OPD for a benign or premalignant pathology in a high-volume center (2011-2021). An electronic three-part questionnaire was sent to eligible patients, including two diseases specific QoL questionnaires (the European Organization for Research and Treatment in Cancer Quality of Life Questionnaire for cancer (QLQ-C30) and a pancreatic cancer module (PAN26) and a body image questionnaire. Patient demographics and postoperative data were collected and compared between LPD and OPD. RESULTS: Among 948 patients who underwent PD (137 LPD, 811 OPD), 170 were eligible and 111 responded (58 LPD and 53 OPD). LPD versus OPD showed no difference in mean age (51 vs. 55 years, p = 0.199) and female gender (40% vs. 45%, p = 0.631), but LPD showed lower BMI (24 vs 26; p = 0.028) and higher preoperative pancreatitis (29% vs 13%; p = 0.041). The postoperative outcome showed similar Clavien-Dindo ≥ III morbidity (19% vs. 23%; p = 0.343) and length of stay (24 vs. 21 days, p = 0.963). After a similar median follow-up (3 vs. 3 years; p = 0.122), LPD vs OPD patients reported higher QoL (QLQ-C30: 49.6 vs 56.3; p = 0.07), better pancreas specific health status score (PAN20: 50.5 vs 55.5; p = 0.002), physical functioning (p = 0.002), and activities limitations (p = 0.02). Scar scores were better after LPD regarding esthetics (p = 0.001), satisfaction (p = 0.04), chronic pain at rest (p = 0.036), moving (p = 0.011) or in daily activities (p = 0.02). There was no difference in digestive symptoms (p = 0.995). CONCLUSION: This monocentric study found improved long-term QoL in patients undergoing LPD, as compared to OPD, for benign and premalignant diseases. These results could be considered when choosing the surgical approach in these patients.

Deleting the ß-catenin degradation domain in mouse hepatocytes drives hepatocellular carcinoma or hepatoblastoma-like tumor growth.

BACKGROUND & AIMS: One-third of hepatocellular carcinomas (HCCs) harbor mutations activating the ß-catenin pathway, predominantly via mutations in the CTNNB1 gene itself. Mouse models of Apc loss-of-function are widely used to mimic ß-catenin-dependent tumorigenesis. Given the low prevalence of APC mutations in human HCCs, we aimed to generate liver tumors through CTNNB1 exon 3 deletion (ßcatΔex3). We then compared ßcatΔex3 liver tumors with liver tumors generated via frameshift in exon 15 of Apc (Apcfs-ex15). METHODS: We used hepatocyte-specific and inducible mouse models generated through either a Cre-Lox or a CRISPR/Cas9 approach using adeno-associated virus vectors. Tumors generated by the Cre-Lox models were phenotypically analyzed using immunohistochemistry and were selected for transcriptomic analysis by RNA-sequencing (RNAseq). Mouse RNAseq data were compared to human RNAseq data (8 normal tissues, 48 HCCs, 9 hepatoblastomas) in an integrative analysis. Tumors generated via CRISPR were analyzed using DNA sequencing and immuno-histochemistry. RESULTS: Mice with CTNNB1 exon 3 deletion in hepatocytes developed liver tumors indistinguishable from Apcfs-ex15 liver tumors. Both Apcfs-ex15 and ßcatΔex3 mouse models induced growth of phenotypically distinct tumors (differentiated or undifferentiated). Integrative analysis of human and mouse tumors showed that differentiated mouse tumors cluster with well-differentiated human CTNNB1-mutated tumors. Conversely, undifferentiated mouse tumors cluster with human mesenchymal hepatoblastomas and harbor activated YAP signaling. CONCLUSION: Apcfs-ex15 and ßcatΔex3 mouse models both induce growth of tumors that are transcriptionally similar to either well-differentiated and ß-catenin-activated human HCCs or mesenchymal hepatoblastomas. LAY SUMMARY: New and easy-to-use transgenic mouse models of primary liver cancers have been generated, with mutations in the gene encoding beta-catenin, which are frequent in both adult and pediatric primary liver cancers. The mice develop both types of cancer, constituting a strong preclinical model.

Calibrated Photoacoustic Spectrometer Based on a Conventional Imaging System for In Vitro Characterization of Contrast Agents.

Photoacoustic (PA) imaging systems are spreading in the biomedical community, and the development of new PA contrast agents is an active area of research. However, PA contrast agents are usually characterized with spectrophotometry or uncalibrated PA imaging systems, leading to partial assessment of their PA efficiency. To enable quantitative PA spectroscopy of contrast agents in vitro with conventional PA imaging systems, we have developed an adapted calibration method. Contrast agents in solution are injected in a dedicated non-scattering tube phantom imaged at different optical wavelengths. The calibration method uses a reference solution of cupric sulfate to simultaneously correct for the spectral energy distribution of excitation light at the tube location and perform a conversion of the tube amplitude in the image from arbitrary to spectroscopic units. The method does not require any precise alignment and provides quantitative PA spectra, even with non-uniform illumination and ultrasound sensitivity. It was implemented on a conventional imaging setup based on a tunable laser operating between 680 nm and 980 nm and a 5 MHz clinical ultrasound array. We demonstrated robust calibrated PA spectroscopy with sample volumes as low as 15 µL of known chromophores and commonly used contrast agents. The validated method will be an essential and accessible tool for the development of new and efficient PA contrast agents by improving their quantitative characterization.

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.

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.

The concomitant loss of APC and HNF4α in adult hepatocytes does not contribute to hepatocarcinogenesis driven by ß-catenin activation.

BACKGROUND & AIMS: Loss of hepatocyte nuclear factor-4α (HNF4α), a critical factor driving liver development and differentiation, is frequently associated with hepatocellular carcinoma (HCC). Our recent data revealed that HNF4α level was decreased in mouse and human HCCs with activated ß-catenin signalling. In addition, increasing HNF4α level by miR-34a inhibition slowed tumour progression of ß-catenin-activated HCC in mice. METHODS: We generated a Hnf4aflox/flox/ Apcflox/flox /TTR-CreERT2 (Hnf4a/Apc∆Hep ) mouse line and evaluated the impact of Hnf4a disruption on HCC development and liver homoeostasis. RESULTS: There was no significant impact of Hnf4a disruption on tumour onset and progression in Apc∆Hep model. However, we observed an unexpected phenotype in 28% of Hnf4a∆Hep mice maintained in a conventional animal facility, which presented disorganized portal triads, characterized by stenosis of the portal vein and increased number and size of hepatic arteries and bile ducts. These abnormal portal structures resemble the human idiopathic non-cirrhotic portal hypertension syndrome. We correlated the presence of portal remodelling with a higher expression of protein and mRNA levels of TGFß and BMP7, a key regulator of the TGFß-dependent endothelial-to-mesenchymal transition. CONCLUSION: These data demonstrate that HNF4α does not play a major role during ß-catenin-driven HCC, thus revealing that the tumour suppressor role of HNF4α is far more complex and dependent probably on its temporal expression and tumour context. However, HNF4α loss in adult hepatocytes could induce abnormal portal structures resembling the human idiopathic non-cirrhotic portal hypertension syndrome, which may result from endothelial- and epithelial-to-mesenchymal transitions.

Dual-acting biofunctionalised scaffolds for applications in regenerative medicine.

Off the shelf scaffolds for replacing ultra-small diameter vascular grafts are valuable for reconstruction of diseased or damaged vessels. The limitations for such grafts include optimal handling with ready availability of varied lengths of grafts, graft patency with the ability to replace the function of active cellular mechanisms and adequate mechanical properties to maintain physicochemical function. We used a well-established, solvent casting method for potential tissue replacement scaffold fabrication with incorporated bioactive molecules, which we have previously explored to confer haemocompatibility. These grafts were tested in-vivo within the abdominal aorta of 10 Wistar rats and the patency was clinically and echographically evaluated. Haemocompatibility and endothelialisation were assessed on explants. Biofunctionalised scaffolds were also grafted subcutaneously and intraperitoneally to evaluate integration, inflammation and angiogenesis reactions. The potential wider applications of this dual acting scaffold were evaluated for its interactions with human dermal fibroblasts as well as bronchial epithelial cells. Physicochemical property evaluation of the functionalised grafts has clarified the mechanical strength and permeability. This study confirmed the microsurgical suturability of tubular grafts and graft patency of functionalized scaffolds. The study demonstrated the potential of a dual acting biofunctionalised scaffold's use for a wide range of tissue engineering applications where micro-porous, yet impermeable scaffolds are needed.

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.

Whole organ volumetric sensing Ultrasound Localization Microscopy for characterization of kidney structure.

Glomeruli are the filtration units of the kidney and their function relies heavily on their microcirculation. Despite its obvious diagnostic importance, an accurate estimation of blood flow in the capillary bundle within glomeruli defies the resolution of conventional imaging modalities. Ultrasound Localization Microscopy (ULM) has demonstrated its ability to image in-vivo deep organs in the body. Recently, the concept of sensing ULM or sULM was introduced to classify individual microbubble behavior based on the expected physiological conditions at the micrometric scale. In the kidney of both rats and humans, it revealed glomerular structures in 2D but was severely limited by planar projection. In this work, we aim to extend sULM in 3D to image the whole organ and in order to perform an accurate characterization of the entire kidney structure. The extension of sULM into the 3D domain allows better localization and more robust tracking. The 3D metrics of velocity and pathway angular shift made glomerular mask possible. This approach facilitated the quantification of glomerular physiological parameter such as an interior traveled distance of approximately 7.5 ± 0.6 microns within the glomerulus. This study introduces a technique that characterize the kidney physiology which can serve as a method to facilite pathology assessment. Furthermore, its potential for clinical relevance could serve as a bridge between research and practical application, leading to innovative diagnostics and improved patient care..

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.

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.

Cardiac characterization of mdx mice using high-resolution doppler echocardiography.

OBJECTIVES: Duchenne muscular dystrophy is an X-linked neuromuscular disorder. The heart is traditionally involved, leading to heart failure. The mdx mouse is a natural animal model of the disease. We conducted a prospective study to analyze left ventricular (LV) function in mdx mice at different ages using high-resolution Doppler echocardiography. METHODS: Echocardiography was performed with a 30-MHz cardiac probe. Wild-type and mdx mice were scanned at 10 and 12 months. We measured the interventricular septal wall thickness, posterior wall thickness, and LV diameter in systole and diastole. The LV shortening fraction, LV ejection fraction, and LV mass were then calculated. RESULTS: At 10 months, the shortening fractions in mdx and wild-type mice were relatively close (29% ± 5% versus 25% ± 4%). We found a significant difference in the posterior wall thickness change (40% ± 12% in mdx versus 28% ± 10% in wild-type; P = .048). The LV mass/body weight ratio was higher in mdx than wild-type mice (3.67 ± 0.25 versus 3.39 ± 0.26; P = .05). At 12 months, the LV mass was elevated in mdx mice compared to wild-type (152 ±16 versus 135 ± 3.7 mg; P = .04). The diastolic posterior wall thickness change was decreased in mdx mice at 12 months compared to wild-type (21% ± 7% versus 33% ± 4%; P = .01). The LV ejection fraction was not statistically different between mdx and wild-type mice (50% ± 6% versus 54% ± 2%). CONCLUSIONS: Ten-month-old mdx mice had a significantly higher posterior wall thickness than wild-type mice, but it was not significant at 12 months. In 12-month-old mdx mice, the posterior wall thickness change was significantly lower, and the LV mass was significantly higher. These findings indicate the role of LV function in the early stages of Duchenne muscular dystrophy.

Motion Rejection and Spectral Unmixing for Accurate Estimation of In Vivo Oxygen Saturation Using Multispectral Optoacoustic Tomography.

Multispectral optoacoustic tomography (MSOT) uniquely enables spatial mapping in high resolution of oxygen saturation (SO2), with potential applications in studying pathological complications and therapy efficacy. MSOT offers seamless integration with ultrasonography, by using a common ultrasound (US) detector array. However, MSOT relies on multiple successive acquisitions of optoacoustic (OA) images at different optical wavelengths and the low frame rate of OA imaging makes the MSOT acquisition sensitive to body/respiratory motion. Moreover, the estimation of SO2 is highly sensitive to noise, and artifacts related to the respiratory motion of the animal were identified as the primary source of noise in MSOT. In this work, we propose a two-step image processing method for SO2 estimation in deep tissues. First, to mitigate motion artifacts, we propose a method of selection of OA images acquired only during the respiratory pause of the animal, using ultrafast ultrasound (US) images acquired immediately after each OA acquisition (US image acquisition duration of 1.4 ms and a total delay of 7 ms). We show that gating is more effective using US images than OA images at different optical wavelengths. Second, we propose a novel method that can estimate directly the SO2 value of a pixel and at the same time evaluate the amount of noise present in that pixel. Hence, the method can efficiently eliminate the pixels dominated by noise from the final SO2 map. Our postprocessing method is shown to outperform conventional methods for SO2 estimation, and the method was validated by in vivo oxygen challenge experiments.

Sensing ultrasound localization microscopy for the visualization of glomeruli in living rats and humans.

BACKGROUND: Estimation of glomerular function is necessary to diagnose kidney diseases. However, the study of glomeruli in the clinic is currently done indirectly through urine and blood tests. A recent imaging technique called Ultrasound Localization Microscopy (ULM) has appeared. It is based on the ability to record continuous movements of individual microbubbles in the bloodstream. Although ULM improved the resolution of vascular imaging up to tenfold, the imaging of the smallest vessels had yet to be reported. METHODS: We acquired ultrasound sequences from living humans and rats and then applied filters to divide the data set into slow-moving and fast-moving microbubbles. We performed a double tracking to highlight and characterize populations of microbubbles with singular behaviors. We decided to call this technique "sensing ULM" (sULM). We used post-mortem micro-CT for side-by-side confirmation in rats. FINDINGS: In this study, we report the observation of microbubbles flowing in the glomeruli in living humans and rats. We present a set of analysis tools to extract quantitative information from individual microbubbles, such as remanence time or normalized distance. INTERPRETATION: As glomeruli play a key role in kidney function, it would be possible that their observation yields a deeper understanding of the kidney. It could also be a tool to diagnose kidney diseases in patients. More generally, it will bring imaging capabilities closer to the functional units of organs, which is a key to understand most diseases, such as cancer, diabetes, or kidney failures. FUNDING: This study was funded by the European Research Council under the European Union Horizon H2020 program (ERC Consolidator grant agreement No 772786-ResolveStroke).

Acute stress induces long-term metabolic, functional, and structural remodeling of the heart.

Takotsubo cardiomyopathy is a stress-induced cardiovascular disease with symptoms comparable to those of an acute coronary syndrome but without coronary obstruction. Takotsubo was initially considered spontaneously reversible, but epidemiological studies revealed significant long-term morbidity and mortality, the reason for which is unknown. Here, we show in a female rodent model that a single pharmacological challenge creates a stress-induced cardiomyopathy similar to Takotsubo. The acute response involves changes in blood and tissue biomarkers and in cardiac in vivo imaging acquired with ultrasound, magnetic resonance and positron emission tomography. Longitudinal follow up using in vivo imaging, histochemistry, protein and proteomics analyses evidences a continued metabolic reprogramming of the heart towards metabolic malfunction, eventually leading to irreversible damage in cardiac function and structure. The results combat the supposed reversibility of Takotsubo, point to dysregulation of glucose metabolic pathways as a main cause of long-term cardiac disease and support early therapeutic management of Takotsubo.

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.

Folate-Targeted Liposomal Formulations Improve Effects of Methotrexate in Murine Collagen-Induced Arthritis.

Methotrexate (MTX) is first-line therapy for the treatment of rheumatoid arthritis (RA), however, its use may be limited by side effects notably post-injection malaise. When patients are intolerant or become unresponsive, second-line or antibody therapy may be indicated. A folate-targeted liposomal formulation of MTX (FL-MTX) is tropic to arthritic paws and prevents the onset of collagen-induced arthritis (CIA) in the mouse. We optimized the drug-to-lipid molar ratio to 0.15 and demonstrated the therapeutic efficacy of this form at 2 mg/kg MTX intraperitoneal (i.p.) twice a week. These improved liposomes were present in inflamed joints in proportion to the degree of swelling of the paw and bone remodeling activity. FL-MTX had lower hepatic and renal elimination of MTX than the free substance. FL-MTX provided equivalent results when given i.p. or subcutaneous (s.c.) and FL-MTX 2 mg/kg (drug/lipid 0.15), twice weekly, was similar to or more effective than 35 mg/kg MTX (same route and schedule) in reducing the incidence and swelling in the murine CIA model. These results suggest that FL-MTX is a more potent nanotherapeutic formulation than free MTX treatment. Its potential benefits for patients may include reduced frequency of treatment and lower overall doses for a given response.

Stiffness increases with myofibroblast content and collagen density in mesenchymal high grade serous ovarian cancer.

Women diagnosed with high-grade serous ovarian cancers (HGSOC) are still likely to exhibit a bad prognosis, particularly when suffering from HGSOC of the Mesenchymal molecular subtype (50% cases). These tumors show a desmoplastic reaction with accumulation of extracellular matrix proteins and high content of cancer-associated fibroblasts. Using patient-derived xenograft mouse models of Mesenchymal and Non-Mesenchymal HGSOC, we show here that HGSOC exhibit distinct stiffness depending on their molecular subtype. Indeed, tumor stiffness strongly correlates with tumor growth in Mesenchymal HGSOC, while Non-Mesenchymal tumors remain soft. Moreover, we observe that tumor stiffening is associated with high stromal content, collagen network remodeling, and MAPK/MEK pathway activation. Furthermore, tumor stiffness accompanies a glycolytic metabolic switch in the epithelial compartment, as expected based on Warburg's effect, but also in stromal cells. This effect is restricted to the central part of stiff Mesenchymal tumors. Indeed, stiff Mesenchymal tumors remain softer at the periphery than at the core, with stromal cells secreting high levels of collagens and showing an OXPHOS metabolism. Thus, our study suggests that tumor stiffness could be at the crossroad of three major processes, i.e. matrix remodeling, MEK activation and stromal metabolic switch that might explain at least in part Mesenchymal HGSOC aggressiveness.

Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment.

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.

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.