RESUMO
Lithium induces nephrogenic diabetes insipidus (NDI) and microcystic chronic kidney disease (CKD). As previous clinical studies suggest that NDI is dose-dependent and CKD is time-dependent, we investigated the effect of low exposition to lithium in a long-term experimental rat model. Rats were fed with a normal diet (control group), with the addition of lithium (Li+ group), or with lithium and amiloride (Li+/Ami group) for 6 months, allowing obtaining low plasma lithium concentrations (0.25 ± 0.06 and 0.43 ± 0.16 mmol/L, respectively). Exposition to low concentrations of plasma lithium levels prevented NDI but not microcystic dilations of kidney tubules, which were identified as collecting ducts (CDs) on immunofluorescent staining. Both hypertrophy, characterized by an increase in the ratio of nuclei per tubular area, and microcystic dilations were observed. The ratio between principal cells and intercalated cells was higher in microcystic than in hypertrophied tubules. There was no correlation between AQP2 messenger RNA levels and cellular remodeling of the CD. Additional amiloride treatment in the Li+/Ami group did not allow consistent morphometric and cellular composition changes compared to the Li+ group. Low exposition to lithium prevented overt NDI but not microcystic dilations of the CD, with differential cellular composition in hypertrophied and microcystic CDs, suggesting different underlying cellular mechanisms.
Assuntos
Amilorida , Aquaporina 2 , Diabetes Insípido Nefrogênico , Modelos Animais de Doenças , Túbulos Renais Coletores , Animais , Diabetes Insípido Nefrogênico/induzido quimicamente , Diabetes Insípido Nefrogênico/prevenção & controle , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/patologia , Túbulos Renais Coletores/metabolismo , Masculino , Ratos , Aquaporina 2/metabolismo , Amilorida/farmacologia , Ratos Wistar , Fatores de Tempo , Insuficiência Renal Crônica/prevenção & controle , Insuficiência Renal Crônica/induzido quimicamente , Lítio/farmacologia , Relação Dose-Resposta a DrogaRESUMO
BACKGROUND: Clinical benefits of atezolizumab plus bevacizumab (atezolizumab-bevacizumab) are observed only in a subset of patients with hepatocellular carcinoma and the development of biomarkers is needed to improve therapeutic strategies. The atezolizumab-bevacizumab response signature (ABRS), assessed by molecular biology profiling techniques, has been shown to be associated with progression-free survival after treatment initiation. The primary objective of our study was to develop an artificial intelligence (AI) model able to estimate ABRS expression directly from histological slides, and to evaluate if model predictions were associated with progression-free survival. METHODS: In this multicentre retrospective study, we developed a model (ABRS-prediction; ABRS-P), which was derived from the previously published clustering-constrained attention multiple instance learning (or CLAM) pipeline. We trained the model fit for regression analysis using a multicentre dataset from The Cancer Genome Atlas (patients treated by surgical resection, n=336). The ABRS-P model was externally validated on two independent series of samples from patients with hepatocellular carcinoma (a surgical resection series, n=225; and a biopsy series, n=157). The predictive value of the model was further tested in a series of biopsy samples from a multicentre cohort of patients with hepatocellular carcinoma treated with atezolizumab-bevacizumab (n=122). All samples in the study were from adults (aged ≥18 years). The validation sets were sampled between Jan 1, 2008, to Jan 1, 2023. For the multicentre validation set, the primary objective was to assess the association of high versus low ABRS-P values, defined relative to cross-validation median split thresholds in the first biopsy series, with progression-free survival after treatment initiation. Finally, we performed spatial transcriptomics and matched prediction heatmaps with in situ expression profiles. FINDINGS: Of the 840 patients sampled, 641 (76%) were male and 199 (24%) were female. Across the development and validation datasets, hepatocellular carcinoma risk factors included alcohol intake, hepatitis B and C virus infections, and non-alcoholic steatohepatitis. Using cross-validation in the development series, the mean Pearson's correlation between ABRS-P values and ABRS score (mean expression of ABRS genes) was r=0·62 (SD 0·09; mean p<0·0001, SD<0·0001). The ABRS-P generalised well on the external validation series (surgical resection series, r=0·60 [95% CI 0·51-0·68], p<0·0001; biopsy series, r=0·53 [0·40-0·63], p<0·0001). In the 122 patients treated with atezolizumab-bevacizumab, those with ABRS-P-high tumours (n=74) showed significantly longer median progression-free survival than those with ABRS-P-low tumours (n=48) after treatment initiation (12 months [95% CI 7-not reached] vs 7 months [4-9]; p=0·014). Spatial transcriptomics showed significantly higher ABRS score, along with upregulation of various other immune effectors, in tumour areas with high ABRS-P values versus areas with low ABRS-P values. INTERPRETATION: Our study indicates that AI applied on hepatocellular carcinoma digital slides is able to serve as a biomarker for progression-free survival in patients treated with atezolizumab-bevacizumab. This approach could be used in the development of inexpensive and fast biomarkers for targeted therapies. The combination of AI heatmaps with spatial transcriptomics provides insight on the molecular features associated with predictions. This methodology could be applied to other cancers or diseases and improve understanding of the biological mechanisms that drive responses to treatments. FUNDING: Institut National du Cancer, Fondation ARC, China Scholarship Council, Ligue Contre le Cancer du Val de Marne, Fondation de l'Avenir, Ipsen, and Fondation Bristol Myers Squibb Pour la Recherche en Immuno-Oncologie.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Adolescente , Adulto , Feminino , Humanos , Masculino , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Inteligência Artificial , Bevacizumab/uso terapêutico , Biomarcadores , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Estudos RetrospectivosRESUMO
RATIONALE: Homologous recombination deficiency (HRD), defined as BRCA1/2 mutation (BRCAmut) or high genomic instability, is used to identify ovarian cancer (OC) patients most likely to benefit from PARP inhibitors. While these tests are useful, they are imperfect. Another approach is to measure the capacity of tumor cells to form RAD51 foci in the presence of DNA damage using an immunofluorescence assay (IF). We aimed to describe for the first time this assay in OC and correlate it to platinum response and BRCAmut. METHODS: Tumor samples were prospectively collected from the randomized CHIVA trial of neoadjuvant platinum +/- nintedanib. IF for RAD51, GMN and gH2AX was performed on FFPE blocks. Tumors were considered RAD51-low if ≤10% of GMN-positive tumor cells had ≥5 RAD51 foci. BRCAmut were identified by NGS. RESULTS: 155 samples were available. RAD51 assay was contributive for 92% of samples and NGS available for 77%. gH2AX foci confirmed the presence of significant basal DNA damage. 54% of samples were considered HRD by RAD51 and presented higher overall response rates to neoadjuvant platinum (P = 0.04) and longer progression-free survival (P = 0.02). In addition, 67% of BRCAmut were HRD by RAD51. Among BRCAmut, RAD51-high tumors seem to harbor poorer response to chemotherapy (P = 0.02). CONCLUSIONS: We evaluated a functional assay of HR competency. OC demonstrate high levels of DNA damage, yet 54% fail to form RAD51 foci. These RAD51-low OC tend to be more sensitive to neoadjuvant platinum. The RAD51 assay also identified a subset of RAD51-high BRCAmut tumors with unexpected poor platinum response.
Assuntos
Neoplasias Ovarianas , Platina , Humanos , Feminino , Platina/uso terapêutico , Recombinação Homóloga , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Dano ao DNA , Proteína BRCA1/genética , Rad51 Recombinase/genéticaRESUMO
Cancer-associated fibroblasts (CAFs) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Previously we described four CAF subtypes with specific molecular and functional features. Here, we have refined our CAF subtype signatures using RNAseq and immunostaining with the goal of defining bioinformatically the phenotypic stromal and tumor epithelial states associated with CAF diversity. We used primary CAF cultures grown from patient PDAC tumors, human data sets (in-house and public, including single-cell analyses), genetically engineered mouse PDAC tissues, and patient-derived xenografts (PDX) grown in mice. We found that CAF subtype RNAseq signatures correlated with immunostaining. Tumors rich in periostin-positive CAFs were significantly associated with shorter overall survival of patients. Periostin-positive CAFs were characterized by high proliferation and protein synthesis rates and low α-smooth muscle actin expression and were found in peri-/pre-tumoral areas. They were associated with highly cellular tumors and with macrophage infiltrates. Podoplanin-positive CAFs were associated with immune-related signatures and recruitment of dendritic cells. Importantly, we showed that the combination of periostin-positive CAFs and podoplanin-positive CAFs was associated with specific tumor microenvironment features in terms of stromal abundance and immune cell infiltrates. Podoplanin-positive CAFs identified an inflammatory CAF (iCAF)-like subset, whereas periostin-positive CAFs were not correlated with the published myofibroblastic CAF (myCAF)/iCAF classification. Taken together, these results suggest that a periostin-positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin-positive CAF is associated with an immune-related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis and are of putative interest for future therapeutic stratification of patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Adenocarcinoma , Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Fibroblastos Associados a Câncer/patologia , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/genética , Neoplasias PancreáticasRESUMO
BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) displaying overexpression of immune gene signatures are likely to be more sensitive to immunotherapy, however, the use of such signatures in clinical settings remains challenging. We thus aimed, using artificial intelligence (AI) on whole-slide digital histological images, to develop models able to predict the activation of 6 immune gene signatures. METHODS: AI models were trained and validated in 2 different series of patients with HCC treated by surgical resection. Gene expression was investigated using RNA sequencing or NanoString technology. Three deep learning approaches were investigated: patch-based, classic MIL and CLAM. Pathological reviewing of the most predictive tissue areas was performed for all gene signatures. RESULTS: The CLAM model showed the best overall performance in the discovery series. Its best-fold areas under the receiver operating characteristic curves (AUCs) for the prediction of tumors with upregulation of the immune gene signatures ranged from 0.78 to 0.91. The different models generalized well in the validation dataset with AUCs ranging from 0.81 to 0.92. Pathological analysis of highly predictive tissue areas showed enrichment in lymphocytes, plasma cells, and neutrophils. CONCLUSION: We have developed and validated AI-based pathology models able to predict the activation of several immune and inflammatory gene signatures. Our approach also provides insights into the morphological features that impact the model predictions. This proof-of-concept study shows that AI-based pathology could represent a novel type of biomarker that will ease the translation of our biological knowledge of HCC into clinical practice. LAY SUMMARY: Immune and inflammatory gene signatures may be associated with increased sensitivity to immunotherapy in patients with advanced hepatocellular carcinoma. In the present study, the use of artificial intelligence-based pathology enabled us to predict the activation of these signatures directly from histology.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Inteligência Artificial , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Curva ROCRESUMO
OBJECTIVES: Polyploidy is a fascinating characteristic of liver parenchyma. Hepatocyte polyploidy depends on the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Which role can be assigned to polyploidy during human hepatocellular carcinoma (HCC) development is still an open question. Here, we investigated whether a specific ploidy spectrum is associated with clinical and molecular features of HCC. DESIGN: Ploidy spectra were determined on surgically resected tissues from patients with HCC as well as healthy control tissues. To define ploidy profiles, a quantitative and qualitative in situ imaging approach was used on paraffin tissue liver sections. RESULTS: We first demonstrated that polyploid hepatocytes are the major components of human liver parenchyma, polyploidy being mainly cellular (binuclear hepatocytes). Across liver lobules, polyploid hepatocytes do not exhibit a specific zonation pattern. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumours. Remarkably, nuclear ploidy is specifically amplified in HCC tumours. In fact, nuclear ploidy is amplified in HCCs harbouring a low degree of differentiation and TP53 mutations. Finally, our results demonstrated that highly polyploid tumours are associated with a poor prognosis. CONCLUSIONS: Our results underline the importance of quantification of cellular and nuclear ploidy spectra during HCC tumorigenesis.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Poliploidia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Estudos de Casos e Controles , Diferenciação Celular/genética , Núcleo Celular/patologia , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Feminino , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto JovemRESUMO
PURPOSE: This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes. METHODS: Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues. RESULTS: We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5 days postconception (dpc) to E13 dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13 dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels. CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/ß-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Oftalmopatias Hereditárias/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA/genética , Epitélio Pigmentado da Retina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Idoso , Animais , Western Blotting , Pré-Escolar , Modelos Animais de Doenças , Oftalmopatias Hereditárias/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Ratos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/crescimento & desenvolvimentoRESUMO
BACKGROUND: Patients with primary immunodeficiency disease (PID) who survive to adulthood and willing to have a child mostly are worried whether their disease affects their fertility and/or pregnancy and also if their child would be predisposed to PID. CASE PRESENTATION: We report the outcome of conception, pregnancy and their management in 9 families with definite diagnosis of PID. A chronic granulomatous disease subject with an uneventful pregnancy developed fungal sacral osteomyelitis few weeks after delivery. A pregnant common variable immunodeficiency disease (CVID) patient with idiopathic thrombocytopenia had platelet count dropped before delivery. A sever neutropenic mother who refused to get IFNγ delivered two healthy children. A CVID case intolerant to IVIg with eclampsia and PTE delivered a baby. Another CVID female gave birth to a baby without being on any treatment since she was not diagnosed with immunodeficiency disease at that time. A healthy girl was implanted via preimplantation gender selection in a family who owned a Wiskott Aldrich-affected son. A family who had two children with Ataxia Telangiectasia used donated oocyte for their 3rd child. Prenatal genetic diagnosis was used to screen the fetus for the impaired BTK and CVID genes detected in sibling and father respectively in 2 separate families. CONCLUSION: Pregnancy in PID patients is more complex than normal population. Because, not only it has the chance of being inherited by the offspring, but also there are some risks for the mother if she has any kind of immunity component defects. So consultation with a clinical geneticist is crucial to choose the best available approach. They also should be observed and followed by a clinical immunologist to take the best possible safe care.
Assuntos
Imunodeficiência de Variável Comum , Complicações do Trabalho de Parto , Complicações na Gravidez , Gravidez de Alto Risco/imunologia , Diagnóstico Pré-Natal/métodos , Saúde Reprodutiva/estatística & dados numéricos , Adulto , Idade de Início , Coeficiente de Natalidade , Criança , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/diagnóstico , Imunodeficiência de Variável Comum/epidemiologia , Feminino , Humanos , Irã (Geográfico)/epidemiologia , Avaliação das Necessidades , Complicações do Trabalho de Parto/diagnóstico , Complicações do Trabalho de Parto/epidemiologia , Complicações do Trabalho de Parto/etiologia , Parto/imunologia , Gravidez , Complicações na Gravidez/diagnóstico , Complicações na Gravidez/epidemiologia , Complicações na Gravidez/etiologia , Cuidado Pré-Natal/métodos , Cuidado Pré-Natal/normas , Sobreviventes/estatística & dados numéricosRESUMO
PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1ß, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Ativação de Macrófagos/efeitos dos fármacos , Microglia/patologia , Retina/patologia , Uveíte/tratamento farmacológico , Uveíte/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anticorpos Neutralizantes/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células , Modelos Animais de Doenças , Humanos , Lipopolissacarídeos , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Testes de Neutralização , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Retina/efeitos dos fármacos , Retina/enzimologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Dent disease (DD) is a hereditary renal disorder characterized by low molecular weight (LMW) proteinuria and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl-/H+exchanger ClC-5 have been identified in patients with DD type 1. ClC-5 is essentially expressed in proximal tubules (PT) where it is thought to play a role in maintaining an efficient endocytosis of LMW proteins. However, the exact pathological roles of ClC-5 in progressive dysfunctions observed in DD type 1 are still unclear. To address this issue, we designed a mouse model carrying the most representative type of ClC-5 missense mutations found in DD patients. These mice showed a characteristic DD type 1 phenotype accompanied by altered endo-lysosomal system and autophagy functions. With ageing, KI mice showed increased renal fibrosis, apoptosis and major changes in cell metabolic functions as already suggested in previous DD models. Furthermore, we made the interesting new discovery that the Lipocalin-2-24p3R pathway might be involved in the progression of the disease. These results suggest a crosstalk between the proximal and distal nephron in the pathogenesis mechanisms involved in DD with an initial PT impairment followed by the Lipocalin-2 internalisation and 24p3R overexpression in more distal segments of the nephron. This first animal model of DD carrying a pathogenic mutation of Clcn5 and our findings pave the way aimed at finding therapeutic strategies to limit the consequences of ClC-5 disruption in patients with DD type 1 developing chronic kidney disease.
RESUMO
BACKGROUND: Patients with mismatch repair-deficient (MMRd) endometrial cancer (EC) can derive great benefit from immune checkpoint inhibitors (ICI). However not all responses and predictors of primary resistance are lacking. METHODS: We compared the immune tumor microenvironment of MMRd EC ICI-responders (Rs) and ICI non-responders (NRs), using spatial multiplexed immune profiling and unsupervised hierarchical clustering analysis. RESULTS: Overall, NRs exhibited drastically lower CD8+, absent terminally differentiated T cells, lack of mature tertiary lymphoid structures and dendritic cells, as well as loss of human leukocyte antigen class I. However, no single marker could predict R versus NR with confidence. Clustering analysis identified a combination of four immune features that demonstrated that accurately predicted ICI response, with a discriminative power of 92%. Finally, 80% of NRs lacked programmed death-ligand 1, however, 60% exhibited another actionable immune checkpoint (T-cell immunoglobulin and mucin containing protein-3, indoleamine 2,3-dioxygenase 1, or lymphocyte activation gene 3). CONCLUSIONS: These findings underscore the potential of immune tumor microenvironment features for identifying patients with MMRd EC and primary resistance to ICI who should be oriented towards trials testing novel immunotherapeutic combinations.
Assuntos
Reparo de Erro de Pareamento de DNA , Neoplasias do Endométrio , Inibidores de Checkpoint Imunológico , Humanos , Feminino , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/imunologia , Neoplasias do Endométrio/genética , Microambiente Tumoral , Pessoa de Meia-Idade , IdosoRESUMO
PURPOSE: This study investigates changes in CD8+ cells, CD8+/Foxp3 ratio, HLA I expression, and immune coregulator density at diagnosis and upon neoadjuvant chemotherapy (NACT), correlating changes with clinical outcomes. EXPERIMENTAL DESIGN: Multiplexed immune profiling and cell clustering analysis were performed on paired matched ovarian cancer samples to characterize the immune tumor microenvironment (iTME) at diagnosis and under NACT in patients enrolled in the CHIVA trial (NCT01583322). RESULTS: Several immune cell (IC) subsets and immune coregulators were quantified pre/post-NACT. At diagnosis, patients with higher CD8+ T cells and HLA I+-enriched tumors were associated with a better outcome. The CD8+/Foxp3+ ratio increased significantly post-NACT in favor of increased immune surveillance, and the influx of CD8+ T cells predicted better outcomes. Clustering analysis stratified pre-NACT tumors into four subsets: high Binf, enriched in B clusters; high Tinf and low Tinf, according to their CD8+ density; and desert clusters. At baseline, these clusters were not correlated with patient outcomes. Under NACT, tumors were segregated into three clusters: high BinfTinf, low Tinf, and desert. The high BinfTinf, more diverse in IC composition encompassing T, B, and NK cells, correlated with improved survival. PDL1 was rarely expressed, whereas TIM3, LAG3, and IDO1 were more prevalent. CONCLUSIONS: Several iTMEs exist during tumor evolution, and the NACT impact on iTME is heterogeneous. Clustering analysis of patients unravels several IC subsets within ovarian cancer and can guide future personalized approaches. Targeting different checkpoints such as TIM3, LAG3, and IDO1, more prevalent than PDL1, could more effectively harness antitumor immunity in this anti-PDL1-resistant malignancy.
Assuntos
Linfócitos T CD8-Positivos , Terapia Neoadjuvante , Neoplasias Ovarianas , Microambiente Tumoral , Humanos , Feminino , Microambiente Tumoral/imunologia , Terapia Neoadjuvante/métodos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/mortalidade , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Pessoa de Meia-Idade , Fatores de Transcrição Forkhead/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Idoso , Adulto , Biomarcadores Tumorais , Receptor Celular 2 do Vírus da Hepatite A/metabolismoRESUMO
Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.
Assuntos
Cromatina/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Hepatócitos/virologia , Interfase , Mitose , Proteínas de Transporte/metabolismo , Linhagem Celular , Nucléolo Celular/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Expressão Gênica , Proteína HMGB2/metabolismo , Hepatócitos/metabolismo , Humanos , Plasmídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Proteínas de Ligação a RNARESUMO
BACKGROUND: Progress in breast cancer (BC) research relies on the availability of suitable cell lines that can be implanted in immunocompetent laboratory mice. The best studied mouse strain, C57BL/6, is also the only one for which multiple genetic variants are available to facilitate the exploration of the cancer-immunity dialog. Driven by the fact that no hormone receptor-positive (HR+) C57BL/6-derived mammary carcinoma cell lines are available, we decided to establish such cell lines. METHODS: BC was induced in female C57BL/6 mice using a synthetic progesterone analog (medroxyprogesterone acetate, MPA) combined with a DNA damaging agent (7,12-dimethylbenz[a]anthracene, DMBA). Cell lines were established from these tumors and selected for dual (estrogen+progesterone) receptor positivity, as well as transplantability into C57BL/6 immunocompetent females. RESULTS: One cell line, which we called B6BC, fulfilled these criteria and allowed for the establishment of invasive estrogen receptor-positive (ER+) tumors with features of epithelial to mesenchymal transition that were abundantly infiltrated by myeloid immune populations but scarcely by T lymphocytes, as determined by single-nucleus RNA sequencing and high-dimensional leukocyte profiling. Such tumors failed to respond to programmed cell death-1 (PD-1) blockade, but reduced their growth on treatment with ER antagonists, as well as with anthracycline-based chemotherapy, which was not influenced by T-cell depletion. Moreover, B6BC-derived tumors reduced their growth on CD11b blockade, indicating tumor sustainment by myeloid cells. The immune environment and treatment responses recapitulated by B6BC-derived tumors diverged from those of ER+ TS/A cell-derived tumors in BALB/C mice, and of ER- E0771 cell-derived and MPA/DMBA-induced tumors in C57BL/6 mice. CONCLUSIONS: B6BC is the first transplantable HR+ BC cell line derived from C57BL/6 mice and B6BC-derived tumors recapitulate the complex tumor microenvironment of locally advanced HR+ BC naturally resistant to PD-1 immunotherapy.
Assuntos
Carcinoma , Progesterona , Camundongos , Feminino , Animais , Transição Epitelial-Mesenquimal , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Primary liver cancer arises either from hepatocytic or biliary lineage cells, giving rise to hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICCA). Combined hepatocellular- cholangiocarcinomas (cHCC-CCA) exhibit equivocal or mixed features of both, causing diagnostic uncertainty and difficulty in determining proper management. Here, we perform a comprehensive deep learning-based phenotyping of multiple cohorts of patients. We show that deep learning can reproduce the diagnosis of HCC vs. CCA with a high performance. We analyze a series of 405 cHCC-CCA patients and demonstrate that the model can reclassify the tumors as HCC or ICCA, and that the predictions are consistent with clinical outcomes, genetic alterations and in situ spatial gene expression profiling. This type of approach could improve treatment decisions and ultimately clinical outcome for patients with rare and biphenotypic cancers such as cHCC-CCA.
Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Colangiocarcinoma , Aprendizado Profundo , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Ductos Biliares Intra-Hepáticos , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Estudos RetrospectivosRESUMO
Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1ß expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1ß expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.
Assuntos
Neovascularização de Coroide/prevenção & controle , Endotélio Vascular/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Macrófagos Peritoneais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Western Blotting , Proteínas de Ligação ao Cálcio , Primers do DNA , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND INFORMATION: Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization. RESULTS: Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-ß-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet. CONCLUSIONS: We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.
Assuntos
Diferenciação Celular , Citosol/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Lipídeos/isolamento & purificação , Proteoma/metabolismo , Animais , Células CACO-2 , Células Cultivadas , Células HeLa , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de ÓrgãosRESUMO
Tumour diagnosis relies on the visual examination of histological slides by pathologists through a microscope eyepiece. Digital pathology, the digitalization of histological slides at high magnification with slides scanners, has raised the opportunity to extract quantitative information due to image analysis. In the last decade, medical image analysis has made exceptional progress due to the development of artificial intelligence (AI) algorithms. AI has been successfully used in the field of medical imaging and more recently in digital pathology. The feasibility and usefulness of AI assisted pathology tasks have been demonstrated in the very last years and we can expect those developments to be applied to routine histopathology in the future. In this review, we will describe and illustrate this technique and present the most recent applications in the field of tumour histopathology. LINKED ARTICLES: This article is part of a themed issue on Molecular imaging - visual themed issue. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.21/issuetoc.
Assuntos
Inteligência Artificial , Neoplasias , Algoritmos , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias/diagnósticoRESUMO
AIM: Type A intercalated cells of the renal collecting duct participate in the maintenance of the acid/base balance through their capacity to adapt proton secretion to homeostatic requirements. We previously showed that increased proton secretion stems in part from the enlargement of the population of proton secreting cells in the outer medullary collecting duct through division of fully differentiated cells, and that this response is triggered by growth/differentiation factor 15. This study aimed at deciphering the mechanism of acid load-induced secretion of Gdf15 and its mechanism of action. METHODS: We developed an original method to evaluate the proliferation of intercalated cells and applied it to genetically modified or pharmacologically treated mice under basal and acid-loaded conditions. RESULTS: Gdf15 is secreted by principal cells of the collecting duct in response to the stimulation of vasopressin receptors. Vasopressin-induced production of cAMP triggers activation of AMP-stimulated kinases and of Na,K-ATPase, and induction of p53 and Gdf15. Gdf15 action on intercalated cells is mediated by ErbB2 receptors, the activation of which triggers the expression of cyclin d1, of p53 and anti-proliferative genes, and of Egr1. CONCLUSION: Acidosis-induced proliferation of intercalated cells results from a cross talk with principal cells which secrete Gdf15 in response to their stimulation by vasopressin. Thus, vasopressin is a major determinant of the collecting duct cellular homeostasis as it promotes proliferation of intercalated cells under acidosis conditions and of principal cells under normal acid-base status.