RESUMEN
The prostate gland consists of basal and luminal cells arranged as pseudostratified epithelium. In tissue recombination models, only basal cells reconstitute a complete prostate gland, yet murine lineage-tracing experiments show that luminal cells generate basal cells. It has remained challenging to address the molecular details of these transitions and whether they apply to humans, due to the lack of culture conditions that recapitulate prostate gland architecture. Here, we describe a 3D culture system that supports long-term expansion of primary mouse and human prostate organoids, composed of fully differentiated CK5+ basal and CK8+ luminal cells. Organoids are genetically stable, reconstitute prostate glands in recombination assays, and can be experimentally manipulated. Single human luminal and basal cells give rise to organoids, yet luminal-cell-derived organoids more closely resemble prostate glands. These data support a luminal multilineage progenitor cell model for prostate tissue and establish a robust, scalable system for mechanistic studies.
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Técnicas de Cultivo de Órganos , Organoides , Próstata/citología , Andrógenos/metabolismo , Humanos , Masculino , Células Madre/citología , Células Madre/metabolismoRESUMEN
Alternative cleavage and polyadenylation (APA) is emerging as an important layer of gene regulation. Factors controlling APA are largely unknown. We developed a reporter-based RNAi screen for APA and identified PABPN1 as a regulator of this process. Genome-wide analysis of APA in human cells showed that loss of PABPN1 resulted in extensive 3' untranslated region shortening. Messenger RNA transcription, stability analyses, and in vitro cleavage assays indicated enhanced usage of proximal cleavage sites (CSs) as the underlying mechanism. Using Cyclin D1 as a test case, we demonstrated that enhanced usage of proximal CSs compromises microRNA-mediated repression. Triplet-repeat expansion in PABPN1 (trePABPN1) causes autosomal-dominant oculopharyngeal muscular dystrophy (OPMD). The expression of trePABPN1 in both a mouse model of OPMD and human cells elicited broad induction of proximal CS usage, linked to binding to endogenous PABPN1 and its sequestration in nuclear aggregates. Our results elucidate a novel function for PABPN1 as a suppressor of APA.
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Proteína II de Unión a Poli(A)/metabolismo , Poliadenilación , Procesamiento Postranscripcional del ARN , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Línea Celular , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Mutación , Proteína II de Unión a Poli(A)/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.
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Aductos de ADN , Neoplasias , Humanos , Carcinógenos/toxicidad , Carcinógenos/metabolismo , Hígado/metabolismo , Organoides/metabolismoRESUMEN
During neoplastic transformation, cells can promote their own growth by activating proto-oncogenes. Reporting in Cell, Iliopoulos et al. (2009) now show that in certain cell types, a transient oncogenic signal is sufficient to induce neoplastic transformation and to maintain it through a positive feedback loop driven by the inflammatory cytokine interleukin-6.
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Transformación Celular Neoplásica , Retroalimentación Fisiológica , Inflamación , Interleucina-6/metabolismo , HumanosRESUMEN
Intratumor heterogeneity is thought to be the driving force of tumor evolution and therapy resistance. Yet tools to study these processes are limited. In this issue, Guernet et al. (2016) devised clustered regularly interspaced short palindromic repeats (CRISPR)-barcoding to functionally annotate specific mutations and study clonal evolution in heterogeneous cell populations.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Oncogenes , Humanos , MutaciónRESUMEN
Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.
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Reparación del ADN/genética , Guanosina/análogos & derivados , Neuroblastoma/genética , Adenina/metabolismo , Niño , Citosina/metabolismo , Daño del ADN , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Femenino , Guanina/metabolismo , Guanosina/genética , Guanosina/metabolismo , Humanos , Masculino , Mutagénesis , Recurrencia Local de Neoplasia/genética , Neuroblastoma/metabolismo , Estrés Oxidativo , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Patient-derived tumour xenografts and tumour organoids have become important preclinical model systems for cancer research. Both models maintain key features from their parental tumours, such as genetic and phenotypic heterogeneity, which allows them to be used for a wide spectrum of applications. In contrast to patient-derived xenografts, organoids can be established and expanded with high efficiency from primary patient material. On the other hand, xenografts retain tumour-stroma interactions, which are known to contribute to tumorigenesis. In this review, we discuss recent advances in patient-derived tumour xenograft and tumour organoid model systems and compare their promises and challenges as preclinical models in cancer research.
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Neoplasias/patología , Organoides/patología , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Humanos , Modelos Biológicos , Organoides/citología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Gene fusions are important cancer drivers in pediatric cancer and their accurate detection is essential for diagnosis and treatment. Clinical decision-making requires high confidence and precision of detection. Recent developments show RNA sequencing (RNA-seq) is promising for genome-wide detection of fusion products but hindered by many false positives that require extensive manual curation and impede discovery of pathogenic fusions. METHODS: We developed Fusion-sq to overcome existing disadvantages of detecting gene fusions. Fusion-sq integrates and "fuses" evidence from RNA-seq and whole genome sequencing (WGS) using intron-exon gene structure to identify tumor-specific protein coding gene fusions. Fusion-sq was then applied to the data generated from a pediatric pan-cancer cohort of 128 patients by WGS and RNA sequencing. RESULTS: In a pediatric pan-cancer cohort of 128 patients, we identified 155 high confidence tumor-specific gene fusions and their underlying structural variants (SVs). This includes all clinically relevant fusions known to be present in this cohort (30 patients). Fusion-sq distinguishes healthy-occurring from tumor-specific fusions and resolves fusions in amplified regions and copy number unstable genomes. A high gene fusion burden is associated with copy number instability. We identified 27 potentially pathogenic fusions involving oncogenes or tumor-suppressor genes characterized by underlying SVs, in some cases leading to expression changes indicative of activating or disruptive effects. CONCLUSIONS: Our results indicate how clinically relevant and potentially pathogenic gene fusions can be identified and their functional effects investigated by combining WGS and RNA-seq. Integrating RNA fusion predictions with underlying SVs advances fusion detection beyond extensive manual filtering. Taken together, we developed a method for identifying candidate gene fusions that is suitable for precision oncology applications. Our method provides multi-omics evidence for assessing the pathogenicity of tumor-specific gene fusions for future clinical decision making.
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Neoplasias , Niño , Humanos , Neoplasias/genética , RNA-Seq , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Medicina de Precisión , Análisis de Secuencia de ARN/métodos , Fusión Génica , Secuenciación Completa del GenomaRESUMEN
This review highlights the role of several immunomodulating elements contributing to the tumor microenvironment of various pediatric renal tumors including Wilms tumor. The roles of innate and adaptive immune cells in renal tumors are summarized as well as immunomodulatory cytokines and other proteins. The expression and the predictive role of checkpoint modulators like PD-L1 and immunomodulating proteins like glypican-3, B7-H3, COX-2 are highlighted with a translational view toward potential therapeutic innovations. We further discuss the current state of preclinical models in advancing this field of study. Finally, examples of clinical trials of immunomodulating strategies such as monoclonal antibodies and chimeric antigen receptor T (CAR-T) cells for relapsed/refractory/progressive pediatric renal tumors are described.
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Neoplasias Renales , Microambiente Tumoral , Niño , Humanos , Antígeno B7-H1 , Neoplasias Renales/tratamiento farmacológico , Inmunomodulación , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
In acute lymphoblastic leukemia (ALL), chromosomal translocations involving the KMT2A gene represent highly unfavorable prognostic factors and most commonly occur in patients less than 1 year of age. Rearrangements of the KMT2A gene drive epigenetic changes that lead to aberrant gene expression profiles that strongly favor leukemia development. Apart from this genetic lesion, the mutational landscape of KMT2A-rearranged ALL is remarkably silent, providing limited insights for the development of targeted therapy. Consequently, identifying potential therapeutic targets often relies on differential gene expression, yet the inhibition of these genes has rarely translated into successful therapeutic strategies. Therefore, we performed CRISPR-Cas9 knock-out screens to search for genetic dependencies in KMT2A-rearranged ALL. We utilized small-guide RNA libraries directed against the entire human epigenome and kinome in various KMT2A-rearranged ALL, as well as wild-type KMT2A ALL cell line models. This screening approach led to the discovery of the epigenetic regulators ARID4B and MBD3, as well as the receptor kinase BMPR2 as novel molecular vulnerabilities and attractive therapeutic targets in KMT2A-rearranged ALL.
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Sistemas CRISPR-Cas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Biblioteca de Genes , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factores de Transcripción , Línea Celular , Antígenos de Neoplasias , Proteínas de NeoplasiasRESUMEN
Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.
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Benzo(a)pireno , Aductos de ADN , Organoides , Humanos , Activación Metabólica , Benzo(a)pireno/toxicidad , Citocromo P-450 CYP1A1/metabolismo , Aductos de ADN/metabolismo , Hígado/metabolismo , Organoides/efectos de los fármacos , Organoides/metabolismoRESUMEN
BACKGROUND: Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists. RESULTS: Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre-tubular aggregates, renal vesicles, and in segments of S-shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single-cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long-term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons. CONCLUSIONS: Given the profound early mesenchymal expression and MET signature of LGR6+ cells, together with the lineage tracing of mesenchymal LGR6+ cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability.
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Nefronas , Células Madre , Diferenciación Celular , Mesodermo , Organogénesis/genéticaRESUMEN
Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Intestinos/patología , Mutación/genética , Organoides/metabolismo , Organoides/patología , Células Madre/patología , Aneuploidia , Animales , Sistemas CRISPR-Cas , Niño , Preescolar , Neoplasias Colorrectales/metabolismo , Femenino , Genes APC , Genes p53/genética , Xenoinjertos , Humanos , Imidazoles , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Persona de Mediana Edad , Mutagénesis Sitio-Dirigida , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Trasplante de Neoplasias , Piperazinas , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína Smad4/deficiencia , Nicho de Células Madre/fisiología , Células Madre/metabolismoRESUMEN
Binding within or nearby target genes involved in cell proliferation and survival enables the p53 tumor suppressor gene to regulate their transcription and cell-cycle progression. Using genome-wide chromatin-binding profiles, we describe binding of p53 also to regions located distantly from any known p53 target gene. Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound enhancer regions (p53BERs) indeed contain enhancer activity and interact intrachromosomally with multiple neighboring genes to convey long-distance p53-dependent transcription regulation. Furthermore, p53BERs produce, in a p53-dependent manner, enhancer RNAs (eRNAs) that are required for efficient transcriptional enhancement of interacting target genes and induction of a p53-dependent cell-cycle arrest. Thus, our results ascribe transcription enhancement activity to p53 with the capacity to regulate multiple genes from a single genomic binding site. Moreover, eRNA production from p53BERs is required for efficient p53 transcription enhancement.
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Elementos de Facilitación Genéticos , ARN/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Puntos de Control del Ciclo Celular/genética , Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes , Humanos , Células MCF-7 , Modelos Genéticos , Unión Proteica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/metabolismoRESUMEN
The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.
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Proliferación Celular , Lesiones Cardíacas/fisiopatología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Relacionadas con la Folistatina/genética , Proteínas Relacionadas con la Folistatina/metabolismo , Lesiones Cardíacas/genética , Lesiones Cardíacas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Embarazo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Adult stem cells from a variety of organs can be expanded long-term in vitro as three-dimensional organotypic structures termed organoids. These adult stem cell-derived organoids retain their organ identity and remain genetically stable over long periods of time. The ability to grow organoids from patient-derived healthy and diseased tissue allows for the study of organ development, tissue homeostasis and disease. In this Review, we discuss the generation of adult stem cell-derived organoid cultures and their applications in in vitro disease modeling, personalized cancer therapy and regenerative medicine.
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Células Madre Adultas/citología , Organoides/citología , Investigación Biomédica Traslacional , Adulto , Humanos , Neoplasias/terapia , Medicina de Precisión , Medicina RegenerativaRESUMEN
Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role in tumor suppression. Intriguingly, similar to lncRNA-OIS1, silencing DPP4 caused senescence bypass, and ectopic expression of DPP4 in lncRNA-OIS1 knockdown cells restored the senescent phenotype. Thus, our data indicate that lncRNA-OIS1 links oncogenic induction and senescence with the activation of the tumor suppressor DPP4.
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Senescencia Celular/genética , Dipeptidil Peptidasa 4/genética , ARN Largo no Codificante/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Expresión Génica , Genes ras , Genoma , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
In the adenoma-carcinoma sequence, it is proposed that intestinal polyps evolve through a set of defined mutations toward metastatic colorectal cancer (CRC). Here, we dissect this adenoma-carcinoma sequence in vivo by using an orthotopic organoid transplantation model of human colon organoids engineered to harbor different CRC mutation combinations. We demonstrate that sequential accumulation of oncogenic mutations in Wnt, EGFR, P53, and TGF-ß signaling pathways facilitates efficient tumor growth, migration, and metastatic colonization. We show that reconstitution of specific niche signals can restore metastatic growth potential of tumor cells lacking one of the oncogenic mutations. Our findings imply that the ability to metastasize-i.e., to colonize distant sites-is the direct consequence of the loss of dependency on specific niche signals.
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Neoplasias Colorrectales/genética , Organoides/trasplante , Adulto , Animales , Movimiento Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/fisiopatología , Modelos Animales de Enfermedad , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Ingeniería Genética , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia/genética , Procesos Neoplásicos , Organoides/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
INTRODUCTION: Angiogenesis is critical for tumor growth and metastasis. Bevacizumab is an antiangiogenic drug used to treat various adult and childhood solid tumors. Its potential efficacy in Wilms tumor (WT) with poor prognosis is not established. AREAS COVERED: The response to bevacizumab-containing regimens in relapsed or refractory WT was reviewed in available literature. Searches were conducted using PubMed, Scopus, and ClinicalTrials.gov databases. Eight papers were identified, published between 2007 and 2020, including six treatment regimens, predominantly vincristine, irinotecan, and bevacizumab (VIB) ± temozolomide (VITB). Among 16 evaluable patients, there were two complete responses, seven partial responses, five patients achieved stable disease (SD), and two patients had progressive disease. Objective responses (OR) were observed in 56% of all cases. OR or SD was observed in 89% (8/9) patients who received VIB/VITB. Bevacizumab was generally well tolerated. Related toxicities included hypertension, proteinuria, and delayed wound healing. EXPERT OPINION: This review suggests potential effectiveness and good tolerability of bevacizumab in the setting of relapsed/refractory WT when used in combination with other drugs. Such combination therapies may serve as a bridging treatment option to other interventions and more personalized treatment options in the future; however, focused trials are needed to obtain additional evidence.
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Inhibidores de la Angiogénesis , Protocolos de Quimioterapia Combinada Antineoplásica , Bevacizumab , Neoplasias Renales , Recurrencia Local de Neoplasia , Tumor de Wilms , Humanos , Bevacizumab/administración & dosificación , Bevacizumab/efectos adversos , Bevacizumab/farmacología , Tumor de Wilms/tratamiento farmacológico , Tumor de Wilms/patología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/efectos adversos , Inhibidores de la Angiogénesis/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Recurrencia Local de Neoplasia/tratamiento farmacológico , Niño , Pronóstico , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patologíaRESUMEN
CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma (DLBCL) patient-derived xenograft (PDX) model. Whereas the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy, rather CD70 interaction in cis with the nanoCAR induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9, resulted in dramatically enhanced functionality in the DLBCL PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knock out (KO) CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that WT CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. On the other hand, the gene signature of KO CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products that led to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.