Multicentre randomised trial of screening with sFlt1/PlGF and planned delivery to prevent pre-eclampsia at term: protocol of the PE37 study.

INTRODUCTION: Pre-eclampsia affects ~5%-7% of pregnancies. Although improved obstetric care has significantly diminished its associated maternal mortality, it remains a leading cause of maternal morbidity and mortality in the world. Term pre-eclampsia accounts for 70% of all cases and a large proportion of maternal-fetal morbidity related to this condition. Unlike in preterm pre-eclampsia, the prediction and prevention of term pre-eclampsia remain unsolved. Previously proposed approaches are based on combined third-trimester screening and/or prophylactic drugs, but these policies are unlikely to be widely implementable in many world settings. Recent evidence shows that the soluble fms-like tyrosine kinase-1 (s-Flt-1) to placental growth factor (PlGF) ratio measured at 35-37 weeks' gestation predicts term pre-eclampsia with an 80% detection rate. Likewise, recent studies demonstrate that induction of labour beyond 37 weeks is safe and well accepted by women. We hypothesise that a single-step universal screening for term pre-eclampsia based on sFlt1/PlGF ratio at 35-37 weeks followed by planned delivery beyond 37 weeks reduces the prevalence of term pre-eclampsia without increasing the caesarean section rates or worsening the neonatal outcomes. METHODS AND ANALYSIS: We propose an open-label randomised clinical trial to evaluate the impact of a screening of term pre-eclampsia with the sFlt-1/PlGF ratio followed by planned delivery in asymptomatic nulliparous women at 35-37 weeks. Women will be assigned 1:1 to revealed (sFlt-1/PlGF known to clinicians) versus concealed (unknown) arms. A cut-off of >90th centile is used to define the high risk of subsequent pre-eclampsia and offer planned delivery from 37 weeks. The efficacy variables will be analysed and compared between groups primarily following an intention-to-treat approach, by ORs and their 95% CI. This value will be computed using a Generalised Linear Mixed Model for binary response (study group as fixed effect and the centre as intercept random effect). ETHICS AND DISSEMINATION: The study is conducted under the principles of Good Clinical Practice. This study was accepted by the Clinical Research Ethics Committee of Hospital Clinic Barcelona on 20 November 2020. Subsequent approval by individual ethical committees and competent authorities was granted. The study results will be published in peer-reviewed journals and disseminated at international conferences. TRIAL REGISTRATION NUMBER: NCT04766866.

Diagnóstico y tratamiento de los quistes hepáticos. Utilidad de los marcadores tumorales intraquísticos (CEA y CA 19.9.) / Diagnosis and treatment of hepatic cysts: Usefulness of intracystic tumor markers (CEA and CA 19.9)

Introducción: El tratamiento de los quistes hepáticos requiere del diagnóstico diferencial de quiste simple hepático (QSH) de la neoplasia mucinosa quística (NMQ) hepática. Las características radiológicas no son patognomónicas. Algunos estudios han sugerido la utilidad de los marcadores tumorales (MKT) intraquísticos. Métodos: Análisis retrospectivo de base de datos prospectiva incluyendo pacientes diagnosticados de QSH sintomático desde el 2003 hasta el 2021. El objetivo del estudio es evaluar los resultados del tratamiento de los QSH sintomáticos y analizar la utilidad de la determinación de «carcinoembryonic antigen» (CEA) y «carbohydrate antigen» CA 19.9 intraquísticos. Resultados: Se incluyeron 50 pacientes tratados por quiste sintomático. En 15 pacientes el primer tratamiento fue el drenaje percutáneo. En 35 pacientes se realizó fenestración laparoscópica. Cuatro pacientes se diagnosticaron de lesiones premalignas/malignas (NMQ, NPIB, linfoma B); tres de ellos requirieron una segunda cirugía tras la fenestración y el diagnóstico anatomopatológico. La mediana de los valores de CEA y CA- 19.9 fue de 196μg/L y 227.321U/mL respectivamente. Los pacientes con lesiones premalignas no tuvieron valores elevados de MKT. El valor predictivo positivo fue del 0% en ambos MKT, y el valor predictivo negativo fue de 89% y 91% respectivamente. Conclusiones: Los valores de CEA y CA 19.9 intraquísticos no permiten distinguir los QSH de las NMH. El tratamiento más resolutivo de los QSH sintomáticos es la fenestración quirúrgica. El análisis anatomopatológico de la pared del quiste posibilita su correcto diagnóstico, permitiendo indicar una reintervención quirúrgica en los casos de NMQ.(AU)

Introduction: To decide treatment of hepatic cysts diagnosis between simple hepatic cyst (SHC) and cystic mucinous neoplasm (CMN). Radiological features are not pathognomonic. Some studies have suggested the utility of intracystic tumor markers. Methods: Retrospective analysis of our prospective database including patients treated due to symptomatic SHC from 2003 to 2021. The aim of the study was to evaluate the results of treatment of symptomatic SHC and the usefulness of the determination of intracystic “carcinoembryonic antigen” (CEA) and “carbohydrate antigen” CA 19.9. Results: Fifty patients diagnosed and treated for symptomatic SHC were included. In 15 patients the first treatment was percutaneous drainage. In 35 patients the first treatment was laparoscopic fenestration. Four patients were diagnosed of premalignant or malignant liver cystic lesions (MCN, IPMN, and lymphoma B); three of them required surgery after initial fenestration and pathological diagnosis. Median CEA and CA 19.9 were 196μg/L and 227.321U/mL, respectively. Patients with malignant or premalignant pathology did not have higher levels of intracystic tumor markers. Positive predictive value was 0% for both markers, and negative predictive value was 89% and 91%, respectively. Conclusion: Values of intracystic tumor markers CEA and CA 19.9 do not allow distinguishing simple cysts from cystic liver neoplasms. The most effective treatment for symptomatic simple liver cysts is surgical fenestration. The pathological analysis of the wall of the cysts enables the correct diagnosis, allowing to indicate a surgical reintervention in cases of hepatic cyst neoplasia.(AU)

Diagnosis and treatment of hepatic cysts. Usefulness of intracystic tumor markers (CEA and CA 19.9.).

INTRODUCTION: To decide treatment of hepatic cysts diagnosis between simple hepatic cyst (SHC) and cystic mucinous neoplasm (CMN). Radiological features are not patognomonic. Some studies have suggested the utility of intracystic tumor markers. METHODS: Retrospective analysis of our prospective database including patients treated due to symptomatic SHC from 2003 to 2021. The aim of the study was to evaluate the results of treatment of symptomatic SHC and the usefulness of the determination of intracystic "carcinoembryonic antigen" (CEA) and "carbohydrate antigen" CA 19.9. RESULTS: 50 patients diagnosed and treated for symptomatic SHC were included. In 15 patients the first treatment was percutaneous drainage. In 35 patients the first treatment was laparoscopic fenestration. Four patients were diagnosed of premalignant or malignant liver cystic lesions (MCN, IPMN, lymphoma B); three of them required surgery after initial fenestration and pathological diagnosis. Median CEA and CA 19-9 were 196 µg/L and 227.321 U/mL respectively. Patients with malignant or premalignant pathology did not have higher levels of intracystic tumor markers. Positive predictive value was 0% for both markers, and negative predictive value was 89% and 91% respectively. CONCLUSION: Values of intracystic tumor markers CEA and CA 19-9 do not allow distinguishing simple cysts from cystic liver neoplasms. The most effective treatment for symptomatic simple liver cysts is surgical fenestration. The pathological analysis of the wall of the cysts enables the correct diagnosis, allowing to indicate a surgical reintervention in cases of hepatic cyst neoplasia.

DPPA3-HIF1α axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype.

Tumor relapse is linked to rapid chemoresistance and represents a bottleneck for cancer therapy success. Engagement of a reduced proliferation state is a non-mutational mechanism exploited by cancer cells to bypass therapy-induced cell death. Through combining functional pulse-chase experiments in engineered cells and transcriptomic analyses, we identify DPPA3 as a master regulator of slow-cycling and chemoresistant phenotype in colorectal cancer (CRC). We find a vicious DPPA3-HIF1α feedback loop that downregulates FOXM1 expression via DNA methylation, thereby delaying cell-cycle progression. Moreover, downregulation of HIF1α partially restores a chemosensitive proliferative phenotype in DPPA3-overexpressing cancer cells. In cohorts of CRC patient samples, DPPA3 overexpression acts as a predictive biomarker of chemotherapeutic resistance that subsequently requires reduction in its expression to allow metastatic outgrowth. Our work demonstrates that slow-cycling cancer cells exploit a DPPA3/HIF1α axis to support tumor persistence under therapeutic stress and provides insights on the molecular regulation of disease progression.

Identifying Cell Differentiation in Colorectal Cancer.

The intestinal epithelium is a rapid self-renewing tissue. Stem cells at the bottom of the crypts first give rise to a proliferative progeny that finally differentiates to a variety of cell types. These terminally differentiated intestinal cells are mostly present in the villi of the intestinal wall and serve as functional units to sustain the main purpose of the organ: food absorption. But for a balance homeostasis, the intestine is composed not only by absorptive enterocytes but also by other cell types such as goblet cells that secrete mucus to lubricate the intestinal lumen, Paneth cells that secrete antimicrobial peptides to control microbiome, and others. Many relevant conditions affecting the intestine including chronic inflammation, Crohn's disease, or cancer can alter the composition of these different functional cell types. As a consequence, they can lose their specialized activity as functional units and further contribute to disease progression and malignancy. Measuring the amount of these different cell populations in the intestine is essential to understand the bases of these diseases and their specific contribution to their malignancy. Interestingly, patient-derived xenograft (PDX) models faithfully recapitulate patients' tumors including the proportion of the different cell lineages present in the original tumor. Here we expose some protocols for evaluating the differentiation of intestinal cells in colorectal tumors.

A Label Retaining System to Capture Slow-Cycling Cancer Cells.

Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. Slow-cycling cancer cells (SCCC) represent a cellular status rather than a cell population present in a minor proportion, even in growing tumors. We have adapted the pulse-chase expression of histone H2B fused to enhanced GFP (H2BeGFP) for labelling and isolating SCCC. SCCC show cancer-initiation potential and enhanced chemoresistance, and present a distinctive nongenetic and cell-autonomous gene expression profile shared across different tumor types. The use of our H2BeGFP pulse-chase method opens the possibility to study live SCCC in any growing tissue either cancerous or normal.

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

TET2 controls chemoresistant slow-cycling cancer cell survival and tumor recurrence.

Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. We have adapted the pulse-chase expression of H2BeGFP for labeling and isolating slow-cycling cancer cells (SCCCs). SCCCs showed cancer initiation potential and enhanced chemoresistance. Cells at this slow-cycling status presented a distinctive nongenetic and cell-autonomous gene expression profile shared across different tumor types. We identified TET2 epigenetic enzyme as a key factor controlling SCCC numbers, survival, and tumor recurrence. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labeled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We have shown the enhanced chemoresistance of SCCCs and revealed 5hmC as a biomarker for their clinical identification and TET2 as a potential drug target for SCCC elimination that could extend patients' survival.

The IMPROVE Guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments).

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information).

Uric Acid Is Protective After Cerebral Ischemia/Reperfusion in Hyperglycemic Mice.

Hyperglycemia at stroke onset is associated with poor long-term clinical outcome in numerous studies. Hyperglycemia induces intracellular acidosis, lipid peroxidation, and peroxynitrite production resulting in the generation of oxidative and nitrosative stress in the ischemic tissue. Here, we studied the effects of acute hyperglycemia on in vivo intercellular adhesion molecule-1 (ICAM-1) expression, neutrophil recruitment, and brain damage after ischemia/reperfusion in mice and tested whether the natural antioxidant uric acid was protective. Hyperglycemia was induced by i.p. administration of dextrose 45 min before transient occlusion of the middle cerebral artery. Magnetic resonance imaging (MRI) was performed at 24 h to measure lesion volume. A group of normoglycemic and hyperglycemic mice received an i.v. injection of micron-sized particles of iron oxide (MPIOs), conjugated with either anti-ICAM-1 antibody or control IgG, followed by T2*w MRI. Neutrophil infiltration was studied by immunofluorescence and flow cytometry. A group of hyperglycemic mice received an i.v. infusion of uric acid (16 mg/kg) or the vehicle starting after 45 min of reperfusion. ICAM-1-targeted MPIOs induced significantly larger MRI contrast-enhancing effects in the ischemic brain of hyperglycemic mice, which also showed more infiltrating neutrophils and larger lesions than normoglycemic mice. Uric acid reduced infarct volume in hyperglycemic mice but it did not prevent vascular ICAM-1 upregulation and did not significantly reduce the number of neutrophils in the ischemic brain tissue. In conclusion, hyperglycemia enhances stroke-induced vascular ICAM-1 and neutrophil infiltration and exacerbates the brain lesion. Uric acid reduces the lesion size after ischemia/reperfusion in hyperglycemic mice.

Tankyrase Inhibition Blocks Wnt/ß-Catenin Pathway and Reverts Resistance to PI3K and AKT Inhibitors in the Treatment of Colorectal Cancer.

PURPOSE: Oncogenic mutations in the KRAS/PI3K/AKT pathway are one of the most frequent alterations in cancer. Although PI3K or AKT inhibitors show promising results in clinical trials, drug resistance frequently emerges. We previously revealed Wnt/ß-catenin signaling hyperactivation as responsible for such resistance in colorectal cancer. Here we investigate Wnt-mediated resistance in patients treated with PI3K or AKT inhibitors in clinical trials and evaluate the efficacy of a new Wnt/tankyrase inhibitor, NVP-TNKS656, to overcome such resistance. EXPERIMENTAL DESIGN: Colorectal cancer patient-derived sphere cultures and mouse tumor xenografts were treated with NVP-TNKS656, in combination with PI3K or AKT inhibitors.We analyzed progression-free survival of patients treated with different PI3K/AKT/mTOR inhibitors in correlation with Wnt/ß-catenin pathway activation, oncogenic mutations, clinicopathological traits, and gene expression patterns in 40 colorectal cancer baseline tumors. RESULTS: Combination with NVP-TNKS656 promoted apoptosis in PI3K or AKT inhibitor-resistant cells with high nuclear ß-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear ß-catenin content and progressed earlier upon PI3K/AKT/mTOR inhibition. Nuclear ß-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not. CONCLUSIONS: High nuclear ß-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear ß-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/ß-catenin inhibition and together with ß-catenin may be predictive biomarkers of drug response providing a rationale to stratify colorectal cancer patients to be treated with PI3K/AKT/mTOR and Wnt/ß-catenin inhibitors.

Immature monocytes recruited to the ischemic mouse brain differentiate into macrophages with features of alternative activation.

Acute stroke induces a local inflammatory reaction causing leukocyte infiltration. Circulating monocytes are recruited to the ischemic brain and become tissue macrophages morphologically indistinguishable from reactive microglia. However, monocytes are a heterogeneous population of cells with different functions. Herein, we investigated the infiltration and fate of the monocyte subsets in a mouse model of focal brain ischemia by permanent occlusion of the distal portion of the middle cerebral artery. We separated two main subtypes of CD11b(hi) monocytes according to their expression of the surface markers Ly6C and CD43. Using adoptive transfer of reporter monocytes and monocyte depletion, we identified the pro-inflammatory Ly6C(hi)CD43(lo)CCR2(+) subset as the predominant monocytes recruited to the ischemic tissue. Monocytes were seen in the leptomeninges from where they entered the cortex along the penetrating arterioles. Four days post-ischemia, they had invaded the infarcted core, where they were often located adjacent to blood vessels. At this time, Iba-1(-) and Iba-1(+) cells in the ischemic tissue incorporated BrdU, but BrdU incorporation was rare in the reporter monocytes. The monocyte phenotype progressively changed by down-regulating Ly6C, up-regulating F4/80, expressing low or intermediate levels of Iba-1, and developing macrophage morphology. Moreover, monocytes progressively acquired the expression of typical markers of alternatively activated macrophages, like arginase-1 and YM-1. Collectively, the results show that stroke mobilized immature pro-inflammatory Ly6C(hi)CD43(lo) monocytes that acutely infiltrated the ischemic tissue reaching the core of the lesion. Monocytes differentiated to macrophages with features of alternative activation suggesting possible roles in tissue repair during the sub-acute phase of stroke.

A caveolin-dependent and PI3K/AKT-independent role of PTEN in ß-catenin transcriptional activity.

Loss of the tumour suppressor PTEN is frequent in human melanoma, results in MAPK activation, suppresses senescence and mediates metastatic behaviour. How PTEN loss mediates these effects is unknown. Here we show that loss of PTEN in epithelial and melanocytic cell lines induces the nuclear localization and transcriptional activation of ß-catenin independent of the PI3K-AKT-GSK3ß axis. The absence of PTEN leads to caveolin-1 (CAV1)-dependent ß-catenin transcriptional modulation in vitro, cooperates with NRAS(Q61K) to initiate melanomagenesis in vivo and induces efficient metastasis formation associated with E-cadherin internalization. The CAV1-ß-catenin axis is mediated by a feedback loop in which ß-catenin represses transcription of miR-199a-5p and miR-203, which suppress the levels of CAV1 mRNA in melanoma cells. These data reveal a mechanism by which loss of PTEN increases CAV1-mediated dissociation of ß-catenin from membranous E-cadherin, which may promote senescence bypass and metastasis.

Neutrophil recruitment to the brain in mouse and human ischemic stroke.

Neutrophils are rapidly recruited in response to local tissue infection or inflammation. Stroke triggers a strong inflammatory reaction but the relevance of neutrophils in the ischemic brain is not fully understood, particularly in the absence of reperfusion. We investigated brain neutrophil recruitment in two murine models of permanent ischemia induced by either cauterization of the distal portion of the middle cerebral artery (c-MCAo) or intraluminal MCA occlusion (il-MCAo), and three fatal cases of human ischemic stroke. Flow cytometry analyses revealed progressive neutrophil recruitment after c-MCAo, lesser neutrophil recruitment following il-MCAo, and absence of neutrophils after sham operation. Confocal microscopy identified neutrophils in the leptomeninges from 6 h after the occlusion, in the cortical basal lamina and cortical Virchow-Robin spaces from 15 h, and also in the cortical brain parenchyma at 24 h. Neutrophils showed signs of activation including histone-3 citrullination, chromatin decondensation, and extracellular projection of DNA and histones suggestive of extracellular trap formation. Perivascular neutrophils were identified within the entire cortical infarction following c-MCAo. After il-MCAo, neutrophils prevailed in the margins but not the center of the cortical infarct, and were intraluminal and less abundant in the striatum. The lack of collaterals to the striatum and a collapsed pial anastomotic network due to brain edema in large hemispheric infarctions could impair neutrophil trafficking in this model. Neutrophil extravasation at the leptomeninges was also detected in the human tissue. We concluded that neutrophils extravasate from the leptomeningeal vessels and can eventually reach the brain in experimental animal models and humans with prolonged arterial occlusion.

Infarct volume prediction using apparent diffusion coefficient maps during middle cerebral artery occlusion and soon after reperfusion in the rat.

Middle cerebral artery occlusion (MCAO) in rodents causes brain infarctions of variable sizes that depend on multiple factors, particularly in models of ischemia/reperfusion. This is a major problem for infarct volume comparisons between different experimental groups since unavoidable variability can induce biases in the results and imposes the use of large number of subjects. MRI can help to minimize these difficulties by ensuring that the severity of ischemia is comparable between groups. Furthermore, several studies showed that infarct volumes can be predicted with MRI data obtained soon after ischemia onset. However, such predictive studies require multiparametric MRI acquisitions that cannot be routinely performed, and data processing using complex algorithms that are often not available. The aim here was to provide a simplified method for infarct volume prediction using apparent diffusion coefficient (ADC) data in a model of transient MCAO in rats. ADC images were obtained before, during MCAO and after 60 min of reperfusion. Probability histograms were generated using ADC data obtained either during MCAO, after reperfusion, or both combined. The results were compared to real infarct volumes, i.e.T2 maps obtained at day 7. Assessment of the performance of the estimations showed better results combining ADC data obtained during occlusion and at reperfusion. Therefore, ADC data alone can provide sufficient information for a reasonable prediction of infarct volume if the MRI information is obtained both during the occlusion and soon after reperfusion. This approach can be used to check whether drug administration after MRI acquisition can change infarct volume prediction.

Wilms' tumor protein induces an epithelial-mesenchymal hybrid differentiation state in clear cell renal cell carcinoma.

The Wilms' tumor transcription factor (WT1) was originally classified as a tumor suppressor, but it is now known to also be associated with cancer progression and poor prognosis in several malignancies. WT1 plays an essential role in orchestrating a developmental process known as mesenchymal-to-epithelial transition (MET) during kidney development, but also induces the reverse process, epithelial-to-mesenchymal transition (EMT) during heart development. WT1 is not expressed in the adult kidney, but shows elevated expression in clear cell renal cell carcinoma (ccRCC). However, the role of WT1 in this disease has not been characterized. In this study, we demonstrate that WT1 is upregulated in ccRCC cells that are deficient in the expression of the von Hippel-Lindau tumor suppressor protein (VHL). We found that WT1 transcriptionally activated Snail, a master transcriptional repressor that is known to induce EMT. Although Snail represses E-cadherin and induces mesenchymal characteristics, we found partial maintenance of E-cadherin and associated epithelial characteristics in kidney cells and ccRCC cells that express WT1, since WT1 upregulates E-cadherin expression and competes with Snail repression. These findings support a novel paradigm in which WT1 induces an epithelial-mesenchymal hybrid transition (EMHT), characterized by Snail up-regulation with E-cadherin maintenance, a tumor cell differentiation state in which cancer cells keep both EMT and MET characteristics which may promote tumor cell plasticity and tumor progression.

A personalized preclinical model to evaluate the metastatic potential of patient-derived colon cancer initiating cells.

PURPOSE: Within the aim of advancing precision oncology, we have generated a collection of patient-derived xenografts (PDX) characterized at the molecular level, and a preclinical model of colon cancer metastasis to evaluate drug-response and tumor progression. EXPERIMENTAL DESIGN: We derived cells from 32 primary colorectal carcinomas and eight liver metastases and generated PDX annotated for their clinical data, gene expression, mutational, and histopathological traits. Six models were injected orthotopically into the cecum wall of NOD-SCID mice in order to evaluate metastasis. Three of them were treated with chemotherapy (oxaliplatin) and three with API2 to target AKT activity. Tumor growth and metastasis progression were analyzed by positron emission tomography (PET). RESULTS: Patient-derived cells generated tumor xenografts that recapitulated the same histopathological and genetic features as the original patients' carcinomas. We show an 87.5% tumor take rate that is one of the highest described for implanted cells derived from colorectal cancer patients. Cecal injection generated primary carcinomas and distant metastases. Oxaliplatin treatment prevented metastasis and API2 reduced tumor growth as evaluated by PET. CONCLUSIONS: Our improved protocol for cancer cell engraftment has allowed us to build a rapidly expanding collection of colorectal PDX, annotated for their clinical data, gene expression, mutational, and histopathological statuses. We have also established a mouse model for metastatic colon cancer with patient-derived cells in order to monitor tumor growth, metastasis evolution, and response to treatment by PET. Our PDX models could become the best preclinical approach through which to validate new biomarkers or investigate the metastatic potential and drug-response of individual patients.

IL-10 deficiency exacerbates the brain inflammatory response to permanent ischemia without preventing resolution of the lesion.

Stroke induces inflammation that can aggravate brain damage. This work examines whether interleukin-10 (IL-10) deficiency exacerbates inflammation and worsens the outcome of permanent middle cerebral artery occlusion (pMCAO). Expression of IL-10 and IL-10 receptor (IL-10R) increased after ischemia. From day 4, reactive astrocytes showed strong IL-10R immunoreactivity. Interleukin-10 knockout (IL-10 KO) mice kept in conventional housing showed more mortality after pMCAO than the wild type (WT). This effect was associated with the presence of signs of colitis in the IL-10 KO mice, suggesting that ongoing systemic inflammation was a confounding factor. In a pathogen-free environment, IL-10 deficiency slightly increased infarct volume and neurologic deficits. Induction of proinflammatory molecules in the IL-10 KO brain was similar to that in the WT 6 hours after ischemia, but was higher at day 4, while differences decreased at day 7. Deficiency of IL-10 promoted the presence of more mature phagocytic cells in the ischemic tissue, and enhanced the expression of M2 markers and the T-cell inhibitory molecule CTLA-4. These findings agree with a role of IL-10 in attenuating local inflammatory reactions, but do not support an essential function of IL-10 in lesion resolution. Upregulation of alternative immunosuppressive molecules after brain ischemia can compensate, at least in part, the absence of IL-10.

A subpopulation of smooth muscle cells, derived from melanocyte-competent precursors, prevents patent ductus arteriosus.

BACKGROUND: Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/ß-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes.