RESUMEN
In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.
Asunto(s)
Modelos Inmunológicos , Neoplasias Experimentales/inmunología , Organoides/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Antígeno B7-H1/inmunología , Técnicas de Cocultivo , Femenino , Humanos , Inmunoterapia , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Organoides/patologíaRESUMEN
Racial disparities affect multiple dimensions of epilepsy care including epilepsy surgery. This study aims to further explore these disparities by determining the utilization of invasive neuromodulation devices according to race and ethnicity in a multicenter study of patients living with focal drug-resistant epilepsy (DRE). We performed a post hoc analysis of the Human Epilepsy Project 2 (HEP2) data. HEP2 is a prospective study of patients living with focal DRE involving 10 sites distributed across the United States. There were no statistical differences in the racial distribution of the study population compared to the US population using census data except for patients reporting more than one race. Of 154 patients enrolled in HEP2, 55 (36%) underwent invasive neuromodulation for DRE management at some point in the course of their epilepsy. Of those, 36 (71%) were patients who identified as White. Patients were significantly less likely to have a device if they identified solely as Black/African American than if they did not (odds ratio = .21, 95% confidence interval = .05-.96, p = .03). Invasive neuromodulation for management of DRE is underutilized in the Black/African American population, indicating a new facet of racial disparities in epilepsy care.
Asunto(s)
Epilepsia Refractaria , Epilepsias Parciales , Disparidades en Atención de Salud , Humanos , Epilepsia Refractaria/terapia , Masculino , Femenino , Epilepsias Parciales/terapia , Epilepsias Parciales/etnología , Disparidades en Atención de Salud/estadística & datos numéricos , Disparidades en Atención de Salud/etnología , Adulto , Estudios Prospectivos , Negro o Afroamericano/estadística & datos numéricos , Persona de Mediana Edad , Estados Unidos , Estimulación Encefálica Profunda/estadística & datos numéricos , Estimulación Encefálica Profunda/métodos , Población Blanca/estadística & datos numéricos , Adulto Joven , AdolescenteRESUMEN
Tumors are often infiltrated by T lymphocytes recognizing either self- or mutated antigens but are generally inactive, although they often show signs of prior clonal expansion. Hypothesizing that this may be due to peripheral tolerance, we formulated nanoparticles containing innate immune stimulants that we found were sufficient to activate self-specific CD8+ T cells and injected them into two different mouse tumor models, B16F10 and MC38. These nanoparticles robustly activated and/or expanded antigen-specific CD8+ tumor-infiltrating T cells, along with a decrease in regulatory CD4+ T cells and an increase in Interleukin-17 producers, resulting in significant tumor growth retardation or elimination and the establishment of immune memory in surviving mice. Furthermore, nanoparticles with modification of stimulating human T cells enabled the robust activation of endogenous T cells in patient-derived tumor organoids. These results indicate that breaking peripheral tolerance without regard to the antigen specificity creates a promising pathway for cancer immunotherapy.
Asunto(s)
Antígenos/inmunología , Inmunidad Innata/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Melanoma Experimental/terapia , Animales , Antígenos/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Línea Celular Tumoral , Humanos , Melanoma Experimental/inmunología , Ratones , Nanopartículas/uso terapéuticoRESUMEN
OBJECTIVES: Drug treatment for children with epilepsy should, ideally, be governed by evidence from adequate and well-controlled clinical studies. However, these studies are difficult to conduct, and so direct evidence supporting the informed use of specific drugs is often lacking. The Research Roundtable for Epilepsy (RRE) met in 2020 to align on an approach to therapy development for focal seizures in children age 1 month <2 years of age. METHODS: The RRE reviewed the regulatory landscape, epidemiology, seizure semiology, antiseizure medicine pharmacology, and safety issues applicable to this population. RESULTS: After reviewing evidence, the conclusion was that pediatric efficacy trials would be impracticable to conduct but a waiver of the regulatory requirement to conduct any study would lead to an absence of information to guide dosing in a critical population. Review of available data and discussion of RRE attendees led to the conclusion that the requirements for extrapolation of efficacy from older children down to infants from age 1 month to <2 years old appeared to be met. After the RRE, the US Food and Drug Administration (FDA) approved brivaracetam for use in children with focal epilepsy above the age of 1 month in August 2021 and lacosamide in October 2021, both based on the principle of extrapolation from data in older children. SIGNIFICANCE: These recommendations should result in more rapid accessibility of antiseizure medications for infants.
Asunto(s)
Epilepsias Parciales , Epilepsia , Adolescente , Anticonvulsivantes/uso terapéutico , Niño , Epilepsias Parciales/tratamiento farmacológico , Epilepsia/tratamiento farmacológico , Humanos , Lactante , Lacosamida/uso terapéutico , Convulsiones/tratamiento farmacológicoRESUMEN
From August 27-28, 2020 the Epilepsy Foundation hosted the Pipeline Conference, exploring emerging issues related to antiepileptic drug and device development. The conference featured epilepsy therapeutic companies and academic laboratories developing drugs for focal epilepsies, innovations for rare and ultra-rare diseases, and devices both in clinical trials and approved for use. In this paper, we outline the virtual presentations by the authors, including novel data from their development pipeline.
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Epilepsias Parciales , Epilepsia , Preparaciones Farmacéuticas , Anticonvulsivantes/uso terapéutico , Epilepsias Parciales/tratamiento farmacológico , Epilepsia/tratamiento farmacológico , HumanosRESUMEN
Accurate seizure forecasting is emerging as a near-term possibility due to recent advancements in machine learning and EEG technology improvements. Large-scale data curation and new data element collection through consumer wearables and digital health tools such as longitudinal seizure diary data has uncovered new possibilities for personalized algorithm development that may be used to predict the likelihood of future seizures. The Epilepsy Foundation recognized the unmet need for development in seizure forecasting following a 2016 survey where an overwhelming majority of respondents across all seizure types and frequencies reported that unpredictability of seizures had the strongest impact on their life while living with or caring for someone living with epilepsy. In early 2021, the Epilepsy Foundation conducted an updated survey among those living with epilepsies and/or their caregivers to better understand the use-cases that best suit the needs of our community as seizure forecast research advances. These results will provide researchers with insight into user-acceptance of using a forecasting tool and incorporation into their daily life. Ultimately, this input from people living with epilepsy and caregivers will provide timely feedback on what the community needs are and ensure researchers and companies first and foremost consider these needs in seizure forecasting tools/product development.
RESUMEN
Cancer cells exhibit hyperactive secretory states that maintain cancer cell viability and remodel the tumor microenvironment. However, the oncogenic signals that heighten secretion remain unclear. Here, we show that p53 loss activates prometastatic secretory vesicle biogenesis in the Golgi. p53 loss up-regulates the expression of a Golgi scaffolding protein, progestin and adipoQ receptor 11 (PAQR11), which recruits an adenosine diphosphate ribosylation factor 1-containing protein complex that loads cargos into secretory vesicles. PAQR11-dependent secretion of a protease, PLAU, prevents anoikis and initiates autocrine activation of a PLAU receptor/signal transducer and activator of transcription-3-dependent pathway that up-regulates PAQR11 expression, thereby completing a feedforward loop that amplifies prometastatic effector protein secretion. Pharmacologic inhibition of PLAU receptor impairs the growth and metastasis of p53-deficient cancers. Blockade of PAQR11-dependent secretion inhibits immunosuppressive processes in the tumor microenvironment. Thus, Golgi reprogramming by p53 loss is a key driver of hypersecretion in cancer.
Asunto(s)
Aparato de Golgi , Proteína p53 Supresora de Tumor , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Aparato de Golgi/metabolismo , Ratones , Transporte de Proteínas , Receptores de Progesterona/metabolismo , Vesículas Secretoras/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide, due in part to its proclivity to metastasize. Identifying novel drivers of invasion and metastasis holds therapeutic potential for the disease. We conducted a gain-of-function invasion screen, which identified two separate hits, IMPAD1 and KDELR2, as robust, independent drivers of lung cancer invasion and metastasis. Given that IMPAD1 and KDELR2 are known to be localized to the ER-Golgi pathway, we studied their common mechanism of driving in vitro invasion and in vivo metastasis and demonstrated that they enhance Golgi-mediated function and secretion. Therapeutically inhibiting matrix metalloproteases (MMPs) suppressed both IMPAD1- and KDELR2-mediated invasion. The hits from this unbiased screen and the mechanistic validation highlight Golgi function as one of the key cellular features altered during invasion and metastasis.
Asunto(s)
Aparato de Golgi/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Proteínas de Transporte Vesicular/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Metaloproteinasas de la Matriz/metabolismo , Invasividad Neoplásica , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Alternative splicing has been shown to causally contribute to the epithelial-mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein-protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Empalme Alternativo/genética , Transición Epitelial-Mesenquimal/genética , Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas de Anclaje a la Quinasa A/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo M/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo M/metabolismo , Xenoinjertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Dominios y Motivos de Interacción de Proteínas , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.
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Adenocarcinoma del Pulmón/metabolismo , Cromosomas Humanos Par 1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenocarcinoma del Pulmón/genética , Animales , Cromosomas Humanos Par 1/genética , Ensayo de Inmunoadsorción Enzimática , Aparato de Golgi/metabolismo , Humanos , Técnicas In Vitro , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Microtomografía por Rayos XRESUMEN
Metastatic lung cancer is the leading cause of cancer-associated mortality worldwide, therefore necessitating novel approaches to identify specific genetic drivers for lung cancer progression and metastasis. We recently performed an in vivo gain-of-function genetic screen to identify driver genes of lung cancer metastasis. In the study reported here, we identify TMEM106B as a primary robust driver of lung cancer metastasis. Ectopic expression of TMEM106B could significantly promote the synthesis of enlarged vesicular lysosomes that are laden with elevated levels of active cathepsins. In a TFEB-dependent manner, TMEM106B could modulate the expression of lysosomal genes of the coordinated lysosomal expression and regulation (CLEAR) pathway in lung cancer cells and patient samples. We also demonstrate that TMEM106B-induced lysosomes undergo calcium-dependent exocytosis, thereby releasing active lysosomal cathepsins necessary for TMEM106B-mediated cancer cell invasion and metastasis in vivo, which could be therapeutically prevented by pharmacological inhibition of cathepsins. Further, in TCGA LUAD data sets, 19% of patients show elevated expression of TMEM106B, which predicts for poor disease-free and overall-survival.
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Adenocarcinoma del Pulmón/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Catepsinas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/mortalidad , Adenocarcinoma del Pulmón/patología , Animales , Antineoplásicos/farmacología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Calcio/metabolismo , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/farmacología , Exocitosis , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso/metabolismo , Pronóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteolisis , Transducción de Señal , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Genetic aberrations driving pro-oncogenic and pro-metastatic activity remain an elusive target in the quest of precision oncology. To identify such drivers, we use an animal model of KRAS-mutant lung adenocarcinoma to perform an in vivo functional screen of 217 genetic aberrations selected from lung cancer genomics datasets. We identify 28 genes whose expression promoted tumor metastasis to the lung in mice. We employ two tools for examining the KRAS-dependence of genes identified from our screen: 1) a human lung cell model containing a regulatable mutant KRAS allele and 2) a lentiviral system permitting co-expression of DNA-barcoded cDNAs with Cre recombinase to activate a mutant KRAS allele in the lungs of mice. Mechanistic evaluation of one gene, GATAD2B, illuminates its role as a dual activity gene, promoting both pro-tumorigenic and pro-metastatic activities in KRAS-mutant lung cancer through interaction with c-MYC and hyperactivation of the c-MYC pathway.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Factores de Transcripción GATA/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma del Pulmón/mortalidad , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/terapia , Animales , Línea Celular Tumoral , Femenino , Factores de Transcripción GATA/antagonistas & inhibidores , Factores de Transcripción GATA/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Integrasas/genética , Integrasas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras , Transducción de Señal , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor cells disseminate early in tumor development making metastasis-prevention strategies difficult. Identifying proteins that promote the outgrowth of disseminated tumor cells may provide opportunities for novel therapeutic strategies. Despite multiple studies demonstrating that the mesenchymal-to-epithelial transition (MET) is critical for metastatic colonization, key regulators that initiate this transition remain unknown. We serially passaged lung metastases from a primary triple negative breast cancer xenograft to the mammary fat pads of recipient mice to enrich for gene expression changes that drive metastasis. An unbiased transcriptomic signature of potential metastatic drivers was generated, and a high throughput gain-of-function screen was performed in vivo to validate candidates. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver. CEACAM5 overproduction enriched for an epithelial gene expression pattern and facilitated tumor outgrowth at metastatic sites. Tissues from patients with metastatic breast cancer confirmed elevated levels of CEACAM5 in lung metastases relative to breast tumors, and an inverse correlation between CEACAM5 and the mesenchymal marker vimentin was demonstrated. Thus, CEACAM5 facilitates tumor outgrowth at metastatic sites by promoting MET, warranting its investigation as a therapeutic target and biomarker of aggressiveness in breast cancer.
RESUMEN
Oncogenic gene fusions drive many human cancers, but tools to more quickly unravel their functional contributions are needed. Here we describe methodology permitting fusion gene construction for functional evaluation. Using this strategy, we engineered the known fusion oncogenes, BCR-ABL1, EML4-ALK, and ETV6-NTRK3, as well as 20 previously uncharacterized fusion genes identified in The Cancer Genome Atlas datasets. In addition to confirming oncogenic activity of the known fusion oncogenes engineered by our construction strategy, we validated five novel fusion genes involving MET, NTRK2, and BRAF kinases that exhibited potent transforming activity and conferred sensitivity to FDA-approved kinase inhibitors. Our fusion construction strategy also enabled domain-function studies of BRAF fusion genes. Our results confirmed other reports that the transforming activity of BRAF fusions results from truncation-mediated loss of inhibitory domains within the N-terminus of the BRAF protein. BRAF mutations residing within this inhibitory region may provide a means for BRAF activation in cancer, therefore we leveraged the modular design of our fusion gene construction methodology to screen N-terminal domain mutations discovered in tumors that are wild-type at the BRAF mutation hotspot, V600. We identified an oncogenic mutation, F247L, whose expression robustly activated the MAPK pathway and sensitized cells to BRAF and MEK inhibitors. When applied broadly, these tools will facilitate rapid fusion gene construction for subsequent functional characterization and translation into personalized treatment strategies. Cancer Res; 77(13); 3502-12. ©2017 AACR.