RESUMEN
BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.
Asunto(s)
Antígeno B7-H1 , Neoplasias , Adulto , Humanos , Niño , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Neoplasias/genética , Linfocitos T CD8-positivos/metabolismo , Biomarcadores de Tumor/genética , Microambiente Tumoral/genética , MutaciónRESUMEN
Brain tumours are the most common solid tumour in children and the leading cause of cancer related death in children. Current treatments include surgery, chemotherapy and radiotherapy. The need for aggressive treatment means many survivors are left with permanent severe disability, physical, intellectual and social. Recent progress in immunotherapy, including genetically engineered T cells with chimeric antigen receptors (CARs) for treating cancer, may provide new avenues to improved outcomes for patients with paediatric brain cancer. In this review we discuss advances in CAR T cell immunotherapy, the major CAR T cell targets that are in clinical and pre-clinical development with a focus on paediatric brain tumours, the paediatric brain tumour microenvironment and strategies used to improve CAR T cell therapy for paediatric tumours.
RESUMEN
Fibrosis is characterized by progressively excessive deposition of matrix components and may lead to organ failure. Transforming growth factor-ß (TGF-ß) is a key cytokine involved in tissue repair and fibrosis. TGF-ß's profibrotic signaling pathways converge at activation of ß-catenin. ß-Catenin is an important transcription cofactor whose function depends on its binding partner. Promoting ß-catenin binding to forkhead box protein O (Foxo) via inhibition of its binding to T-cell factor (TCF) reduces kidney fibrosis in experimental murine models. Herein, we investigated whether ß-catenin/Foxo diverts TGF-ß signaling from profibrotic to physiological epithelial healing. In an in vitro model of wound healing (scratch assay), and in an in vivo model of kidney injury, unilateral renal ischemia reperfusion, TGF-ß treatment in combination with either ICG-001 or iCRT3 (ß-catenin/TCF inhibitors) increased ß-catenin/Foxo interaction, increased scratch closure by increased cell proliferation and migration, reduced the TGF-ß-induced mesenchymal differentiation, and healed the ischemia reperfusion injury with less fibrosis. In addition, administration of ICG-001 or iCRT3 reduced the contractile activity induced by TGF-ß in C1.1 cells. Together, our results indicate that redirection of ß-catenin binding from TCF to Foxo promotes ß-catenin/Foxo-mediated epithelial repair. Targeting ß-catenin/Foxo may rebuild normal structure of injured kidney.
Asunto(s)
Proteína Forkhead Box O1/metabolismo , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Transducción de Señal/fisiología , Cicatrización de Heridas/fisiología , beta Catenina/metabolismo , Animales , Fibrosis , RatonesRESUMEN
ß-Catenin is an important co-factor which binds multiple transcriptional molecules and mediates fibrogenic signaling pathways. Its role in kidney transplantation is unknown. We quantified binding of ß-catenin within renal tubular epithelial cells to transcription factors, TCF1 and FoxO1, using a proximity ligation assay in 240 transplanted kidneys, and evaluated their pathological and clinical outcomes. ß-Catenin/FoxO1 binding in 1-month protocol biopsies inversely correlated with contemporaneous chronic fibrosis, subsequent inflammation. and inflammatory fibrosis (P < .001). The relative binding of ß-catenin/TCF1 versus ß-catenin/FoxO1 (TF ratio) was the optimal biomarker, and abnormal in diverse fibrotic transplant diseases. A high 1-month TF ratio was followed by greater tubular atrophy and interstitial fibrosis scores, cortical inflammation, renal impairment, and proteinuria at 1 year (n = 131, all P < .001). The TF ratio was associated with reduced eGFR (AUC 0.817), mild fibrosis (AUC 0.717), and moderate fibrosis (AUC 0.769) using receiver operating characteristic analysis. An independent validation cohort (n = 76) confirmed 1-month TF was associated with 12-month moderate fibrosis (15.8% vs. 2.6%, P = .047), however, not with other outcomes or 10-year graft survival, which limits generalizabilty of these findings. In summary, differential binding of ß-catenin to TCF1 rather than FoxO1 in renal tubular cells was associated with the fibrogenic response in transplanted kidneys.
Asunto(s)
Enfermedades Renales , beta Catenina , Células Epiteliales , Fibrosis , Proteína Forkhead Box O1 , Factor Nuclear 1-alfa del Hepatocito , Humanos , Riñón/patología , Enfermedades Renales/patología , Túbulos Renales/patologíaRESUMEN
An amendment to this paper has been published and can be accessed via the original article.
RESUMEN
Transforming growth factor ß (TGF-ß) is the key cytokine involved in causing fibrosis through cross-talk with major profibrotic pathways. However, inhibition of TGF-ß to prevent fibrosis would also abrogate its anti-inflammatory and wound-healing effects. ß-catenin is a common co-factor in most TGF-ß signaling pathways. ß-catenin binds to T-cell factor (TCF) to activate profibrotic genes and binds to Forkhead box O (Foxo) to promote cell survival under oxidative stress. Using a proximity ligation assay in human kidney biopsies, we found that ß-catenin/Foxo interactions were higher in kidney with little fibrosis, whereas ß-catenin/TCF interactions were upregulated in the kidney of patients with fibrosis. We hypothesised that ß-catenin/Foxo is protective against kidney fibrosis. We found that Foxo1 protected against rhTGF-ß1-induced profibrotic protein expression using a CRISPR/cas9 knockout of Foxo1 or TCF1 in murine kidney tubular epithelial C1.1 cells. Co-administration of TGF-ß with a small molecule inhibitor of ß-catenin/TCF (ICG-001), protected against kidney fibrosis in unilateral ureteral obstruction. Collectively, our human, animal and in vitro findings suggest ß-catenin/Foxo as a therapeutic target in kidney fibrosis.
Asunto(s)
Proteína Forkhead Box O1/metabolismo , Enfermedades Renales/metabolismo , Riñón/metabolismo , beta Catenina/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular , Modelos Animales de Enfermedad , Fibrosis , Proteína Forkhead Box O1/deficiencia , Proteína Forkhead Box O1/genética , Técnicas de Inactivación de Genes , Factor Nuclear 1-alfa del Hepatocito/deficiencia , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Humanos , Riñón/efectos de los fármacos , Riñón/patología , Enfermedades Renales/patología , Enfermedades Renales/prevención & control , Masculino , Ratones , Pirimidinonas/farmacología , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , beta Catenina/antagonistas & inhibidoresRESUMEN
Dimethyl fumarate is an FDA-approved oral immunomodulatory drug with anti-inflammatory properties that induces the upregulation of the anti-oxidant transcription factor, nuclear factor erythroid-derived factor 2. The aim of this study was to determine the efficacy of dimethyl fumarate on interstitial inflammation and renal cyst growth in a preclinical model of nephronophthisis. Four-week-old female Lewis polycystic kidney disease (a genetic ortholog of human nephronophthisis-9) rats received vehicle (V), 10 mg/kg (D10) or 30 mg/kg (D30) ( n = 8-9 each) dimethyl fumarate in drinking water for eight weeks. Age-matched Lewis control rats were also studied ( n = 4 each). Nuclear factor erythroid-derived factor 2 was quantified by whole-slide image analysis of kidney sections. Renal nuclear factor erythroid-derived factor 2 activation was partially reduced in vehicle-treated Lewis polycystic kidney disease rats compared to Lewis control (21.4 ± 1.7 vs. 27.0 ± 1.6%, mean ± SD; P < 0.01). Dimethyl fumarate upregulated nuclear factor erythroid-derived factor 2 in both Lewis Polycystic Kidney Disease (D10: 35.9 ± 3.8; D30: 33.6 ± 3.4%) and Lewis rats (D30: 34.4 ± 1.3%) compared to vehicle-treated rats ( P < 0.05). Dimethyl fumarate significantly reduced CD68+ cell accumulation in Lewis polycystic kidney disease rats (V: 31.7 ± 2.4; D10: 23.0 ± 1.1; D30: 21.5 ± 1.9; P < 0.05). In Lewis polycystic kidney disease rats, dimethyl fumarate did not alter the progression of kidney enlargement (V: 6.4 ± 1.6; D10: 6.9 ± 1.2; D30: 7.3 ± 1.3%) and the percentage cystic index (V: 59.1 ± 2.7; D10: 55.7 ± 3.5; D30: 58.4 ± 2.9%). Renal dysfunction, as determined by the serum creatinine (Lewis + V: 26 ± 4 vs. LPK + V: 60 ± 25 P < 0.01; LPK + D10: 47 ± 7; LPK + D30: 47 ± 9 µmol/L), and proteinuria were also unaffected by dimethyl fumarate treatment. In conclusion, the upregulation of nuclear factor erythroid-derived factor 2 by dimethyl fumarate reduced renal macrophage infiltration in nephronophthisis without adverse effects, suggesting that it could potentially be used in combination with other therapies that reduce the rate of renal cyst growth. Impact statement This is the first study to investigate the effects of dimethyl fumarate in a model of cystic kidney disease. The study assessed the therapeutic efficacy of dimethyl fumarate in upregulating renal nuclear factor erythroid-derived factor 2 expression, reducing macrophage accumulation and cyst progression in a Lewis polycystic kidney disease rat model. This study demonstrates that dimethyl fumarate significantly upregulated renal nuclear factor erythroid-derived factor 2 expression and attenuates renal macrophage infiltration, but had no effect on renal cyst progression, cardiac enlargement, and improving renal function.
Asunto(s)
Antiinflamatorios/uso terapéutico , Dimetilfumarato/uso terapéutico , Inmunosupresores/uso terapéutico , Factor de Transcripción NF-E2/metabolismo , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Animales , Animales Modificados Genéticamente , Cardiomegalia/tratamiento farmacológico , Creatinina/sangre , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Activación Enzimática/efectos de los fármacos , Femenino , Riñón/patología , Macrófagos/inmunología , Factor de Transcripción NF-E2/biosíntesis , FN-kappa B/biosíntesis , Enfermedades Renales Poliquísticas/genética , Proteinuria/sangre , Ratas , Ratas Endogámicas LewRESUMEN
TGF-ß is a key profibrotic factor, but targeting TGF-ß to prevent fibrosis also abolishes its protective anti-inflammatory effects. Here, we investigated the hypothesis that we can redirect TGF-ß signaling by preventing downstream profibrotic interaction of ß-catenin with T cell factor (TCF), thereby enhancing the interaction of ß-catenin with Foxo, a transcription factor that controls differentiation of TGF-ß induced regulatory T cells (iTregs), and thus, enhance anti-inflammatory effects of TGF-ß In iTregs derived from EL4 T cells treated with recombinant human TGF-ß1 (rhTGF-ß1) in vitro, inhibition of ß-catenin/TCF transcription with ICG-001 increased Foxp3 expression, interaction of ß-catenin and Foxo1, binding of Foxo1 to the Foxp3 promoter, and Foxo transcriptional activity. Moreover, the level of ß-catenin expression positively correlated with the level of Foxo1 binding to the Foxp3 promoter and Foxo transcriptional activity. T cell fate mapping in Foxp3gfp Ly5.1/5.2 mice revealed that coadministration of rhTGF-ß1 and ICG-001 further enhanced the expansion of iTregs and natural Tregs observed with rhTGF-ß1 treatment alone. Coadministration of rhTGF-ß1 with ICG-001 also increased the number of Tregs and reduced inflammation and fibrosis in the kidney fibrosis models of unilateral ureteric obstruction and ischemia-reperfusion injury. Notably, ICG-001 prevented the fibrosis in distant organs (lung and liver) caused by rhTGF-ß1. Together, our results show that diversion of ß-catenin from TCF- to Foxo-mediated transcription inhibits the ß-catenin/TCF-mediated profibrotic effects of TGF-ß while enhancing the ß-catenin/Foxo-mediated anti-inflammatory effects. Targeting ß-catenin/Foxo may be a novel therapeutic strategy in the treatment of fibrotic diseases that lead to organ failure.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Riñón/patología , Transducción de Señal , Linfocitos T Reguladores/metabolismo , Factores de Transcripción TCF/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Obstrucción Ureteral/patología , beta Catenina/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular , Citocinas/sangre , Fibrosis , Proteína Forkhead Box O1/metabolismo , Factores de Transcripción Forkhead/genética , Inflamación/patología , Masculino , Ratones , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Pirimidinonas/farmacología , Proteínas Recombinantes/farmacología , Proteína smad3/genética , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/patología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
BACKGROUND: Endothelial-mesenchymal transition (EndoMT) is a major source of myofibroblast formation in kidney fibrosis. Our previous study showed a profibrotic role for matrix metalloproteinase 9 (MMP-9) in kidney fibrosis via induction of epithelial-mesenchymal transition (EMT). Inhibition of MMP-9 activity reduced kidney fibrosis in murine unilateral ureteral obstruction. This study investigated whether MMP-9 also plays a role in EndoMT in human glomerular endothelial cells. RESULTS: TGF-ß1 (10 or 20 ng/ml) induced EndoMT in HKGECs as shown by morphological changes. In addition, VE-cadherin and CD31 were significantly downregulated, whereas α-SMA, vimentin, and N-cadherin were upregulated. RT-PCR revealed that Snail, a known inducer of EMT, was upregulated. The MMP inhibitor GM6001 abrogated TGF-ß1-induced EndoMT. Zymography indicated that MMP-9 was also upregulated in TGF-ß1-treated HKGECs. Recombinant MMP-9 (2 µg/ml) induced EndoMT in HKGECs via Notch signaling, as evidenced by increased formation of the Notch intracellular domain (NICD) and decreased Notch 1. Inhibition of MMP-9 activity by its inhibitor showed a dose-dependent response in preventing TGF-ß1-induced α-SMA and NICD in HKGECs, whereas inhibition of Notch signaling by γ-secretase inhibitor (GSI) blocked rMMP-9-induced EndoMT. CONCLUSIONS: Taken together, our results demonstrate that MMP-9 plays an important role in TGF-ß1-induced EndoMT via upregulation of Notch signaling in HKGECs.
Asunto(s)
Células Endoteliales/metabolismo , Glomérulos Renales/citología , Metaloproteinasa 9 de la Matriz/metabolismo , Mesodermo/citología , Receptores Notch/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Dipéptidos/farmacología , Células Endoteliales/efectos de los fármacos , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Mesodermo/efectos de los fármacos , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
TGF-ß1 induces epithelial-mesenchymal transition (EMT) and autophagy in a variety of cells. However, the role of autophagy in TGF-ß1-induced EMT has not been clearly elucidated and the underlying mechanisms are unclear. In the present study, we found that TGF-ß1 induced both autophagy and EMT in mouse tubular epithelial C1.1 cells. Inhibition of autophagy by 3-methyladenine or siRNA knockdown of Beclin 1 reduced TGF-ß1-induced increase of vimentin and decreased E-cadherin expression. In contrast, rapamycin-associated enhancement of TGF-ß1-induced autophagy increased EMT of C1.1 cells. Serum rescue inhibited autophagy followed by reversal of EMT. Blocking of autophagosome-lysosomal but not proteosomal degradation reduced the decrease of E-cadherin, demonstrating a role for autophagy in degradation of E-cadherin during EMT. Autophagy promoted the activation of Src and Src-associated phosphorylation of ß-catenin at Y-654 leading to pY654-ß-catenin/p-Smad2 complex formation. Chromatin immunoprecipitation assay demonstrated binding by the pY654-ß-catenin/p-Smad2 complex to ILK promoter thus increasing ILK expression. Taken together, our results demonstrate that TGF-ß1-induced autophagy links ß-catenin and Smad signaling to promote EMT in C1.1 cells through a novel pY654-ß-catenin/p-Smad2/ILK pathway. The pathway delineated links disruption of E-cadherin/ß-catenin-mediated cell-cell contact to induction of EMT via upregulation of ILK.
Asunto(s)
Autofagia/fisiología , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Túbulos Renales/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteína Smad2/metabolismo , Regulación hacia Arriba/fisiología , beta Catenina/metabolismo , Animales , Línea Celular , Células Epiteliales/citología , Túbulos Renales/citología , Ratones , Proteínas Serina-Treonina Quinasas/genética , Proteína Smad2/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , beta Catenina/genéticaRESUMEN
BACKGROUND: Hearts from older donors are increasingly being referred for transplantation. However, these hearts are more susceptible to ischemia-reperfusion injury (IRI), reflected in higher rates of primary graft dysfunction. We assessed a strategy of pharmacologic conditioning, supplementing Celsior (Genzyme, Naarden, The Netherlands) preservation solution with glyceryl trinitrate (GTN; Hospira Australia Pty, Ltd, Mulgrave, VIC, Australia), erythropoietin (EPO; Eprex; Janssen-Cilag, North Ryde, NSW, Australia), and zoniporide (ZON; Pfizer, Inc., Groton, CT), to protect older hearts against IRI and improve graft function. METHODS: Wistar rats, aged 3, 12, and 18 months old, were used to represent adolescent, 30-year-old, and 45-year-old human donors, respectively. Animals were subjected to brain death (BD) and hearts stored for 6 hours at 2° to 3°C in Celsior or Celsior supplemented with GTN+EPO+ZON. Cardiac function and lactate dehydrogenase before and after storage were assessed during ex vivo perfusion. Western blots and histopathology were also analyzed. RESULTS: After BD, 18-month hearts demonstrated impaired aortic flow, coronary flow, and cardiac output compared with 3-month hearts (p < 0.001 to p < 0.0001). After storage in Celsior, the recovery of aortic flow, coronary flow, and cardiac output in 18-month BD hearts was further impaired (p < 0.01 vs 3-month hearts). Percentage functional recovery of 18-month BD hearts stored in Celsior supplemented with GTN+EPO+ZON was equivalent to that of 3-month hearts and significantly improved compared with 18-month hearts stored in Celsior alone (p < 0.01 to p < 0.001), with reduced lactate dehydrogenase release (p < 0.01) and myocardial edema (p < 0.05) and elevated phosphorylated extracellular signal-related kinase 1/2 (p < 0.05) and phosphorylated Akt (p < 0.01). CONCLUSIONS: Older hearts are more susceptible to IRI induced by BD and prolonged hypothermic storage. Supplemented Celsior activates cell survival signaling in older hearts, reduces IRI, and enhances donor heart preservation.
Asunto(s)
Corazón , Animales , Trasplante de Corazón , Preservación de Órganos , Soluciones Preservantes de Órganos , Ratas , Ratas WistarRESUMEN
Heterocyclic dithiocarbamates have anti-inflammatory and anti-proliferative effects in rodent models of chronic kidney disease. In this study, we tested the hypothesis that pyrrolidine dithiocarbamate (PDTC) reduces the progression of polycystic kidney disease (PKD). Male Lewis polycystic kidney (LPK) rats (an ortholog of Nek8/NPHP9) received intraperitoneal injections of either saline vehicle or PDTC (40 mg/kg once or twice daily) from postnatal weeks 4 until 11. By serial magnetic resonance imaging at weeks 5 and 10, the relative within-rat increase in total kidney volume and cyst volume were 1.3-fold (P = 0.01) and 1.4-fold (P < 0.01) greater, respectively, in LPK + Vehicle compared to the LPK + PDTC(40 mg/kg twice daily) group. At week 11 in LPK rats, PDTC attenuated the increase in kidney weight to body weight ratio by 25% (P < 0.01) and proteinuria by 66% (P < 0.05 vs. LPK + Vehicle) but did not improve renal dysfunction. By quantitative whole-slide image analysis, PDTC did not alter interstitial CD68+ cell accumulation, interstitial fibrosis, or renal cell proliferation in LPK rats at week 11. The phosphorylated form of the nuclear factor (NF)-κB subunit, p105, was increased in cystic epithelial cells of LPK rats, but was not altered by PDTC. Moreover, PDTC did not significantly alter nuclear expression of the p50 subunit or NF-κB (p65)-DNA binding. Kidney enlargement in LPK rats was resistant to chronic treatment with a proteasome inhibitor, bortezomib. In conclusion, PDTC reduced renal cystic enlargement and proteinuria but lacked anti-inflammatory effects in LPK rats.