RESUMEN
Extracellular vesicles (EVs) are secreted, cell-derived, membrane-bound compartments implicated in various diseases for their ability to influence distant targets and as carriers of biomarkers. Here, we present a protocol for separating EVs from mammalian pancreatic cancer cells and their characterization using western blot and electron microscopy. We then demonstrate how they are utilized to affect tumor development in a murine model of metastatic pancreatic cancer including a method to quantify hepatic tumor burden in histologic samples. For complete details on the use and execution of this protocol, please refer to Dudgeon et al.1.
RESUMEN
The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor due to changes in cell plasticity governed by a distinct transcriptome. Therapeutic strategies that target this distinct biology are needed. We detect an upregulation of the neuronal axon guidance molecule Netrin-1 in PDAC liver metastases that signals through its dependence receptor (DR), uncoordinated-5b (Unc5b), to facilitate metastasis in vitro and in vivo. The mechanism of Netrin-1 induction involves a feedforward loop whereby Netrin-1 on the surface of PDAC-secreted extracellular vesicles prepares the metastatic niche by inducing hepatic stellate cell activation and retinoic acid secretion that in turn upregulates Netrin-1 in disseminated tumor cells via RAR/RXR and Elf3 signaling. While this mechanism promotes PDAC liver metastasis, it also identifies a therapeutic vulnerability, as it can be targeted using anti-Netrin-1 therapy to inhibit metastasis using the Unc5b DR cell death mechanism.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Humanos , Netrina-1 , Retinoides , Células Estrelladas Hepáticas/metabolismo , Línea Celular Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Neoplasias Hepáticas/metabolismo , Receptores de Netrina , Proteínas de Unión al ADN , Factores de Transcripción , Proteínas Proto-Oncogénicas c-etsRESUMEN
Netrin-1 is upregulated in a large fraction of human neoplasms. In multiple animal models, interference with netrin-1 is associated with inhibition of tumor growth and metastasis. Although netrin-1 upregulation was initially described in cancer cells, we report here that in the human colorectal cancer database, the expression of netrin-1 and its receptor UNC5B correlates with a cancer-associated fibroblasts (CAF) signature. Both colon and lung CAF secreted netrin-1 when cocultured with respective cancer cells, and netrin-1 upregulation in CAF was associated with increased cancer cell stemness. Pharmacologic inhibition of netrin-1 with a netrin-1-mAb (Net1-mAb) abrogated the CAF-mediated increase of cancer stemness both in coculture experiments and in mice. Net-1-mAb inhibited intercellular signaling between CAF and cancer cells by modulating CAF-mediated expression of cytokines such as IL6. Together these data demonstrate that netrin-1 is upregulated not only in cancer cells but also in cancer-associated stromal cells. In addition to its direct activity on cancer cells, inhibition of netrin-1 may reduce proneoplastic CAF-cancer cell cross-talk, thus inhibiting cancer plasticity. SIGNIFICANCE: Netrin-1, a navigation cue during embryonic development, is upregulated in cancer-associated fibroblasts and regulates cancer cell stemness.
Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias del Colon/patología , Neoplasias Pulmonares/patología , Netrina-1/biosíntesis , Células A549 , Animales , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Plasticidad de la Célula/fisiología , Neoplasias del Colon/metabolismo , Femenino , Células HCT116 , Xenoinjertos , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones SCID , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores de Netrina/biosíntesis , Regulación hacia ArribaRESUMEN
The microenvironment influences cancer drug response and sustains resistance to therapies targeting receptor-tyrosine kinases. However, if and how the tumor microenvironment can be altered during treatment, contributing to resistance onset, is not known. We show that, under prolonged treatment with tyrosine kinase inhibitors (TKIs), EGFR- or MET-addicted cancer cells displayed a metabolic shift toward increased glycolysis and lactate production. We identified secreted lactate as the key molecule instructing cancer-associated fibroblasts to produce hepatocyte growth factor (HGF) in a nuclear factor κB-dependent manner. Increased HGF, activating MET-dependent signaling in cancer cells, sustained resistance to TKIs. Functional or pharmacological targeting of molecules involved in the lactate axis abrogated in vivo resistance, demonstrating the crucial role of this metabolite in the adaptive process. This adaptive resistance mechanism was observed in lung cancer patients progressed on EGFR TKIs, demonstrating the clinical relevance of our findings and opening novel scenarios in the challenge to drug resistance.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Glucólisis/efectos de los fármacos , Ácido Láctico/metabolismo , Neoplasias Pulmonares , Microambiente Tumoral/efectos de los fármacos , Animales , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica , Receptores ErbB/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones Endogámicos NOD , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The National Association of Medical Examiners commissioned an ad hoc committee to provide recommendations for the investigation, examination, and reporting of deaths in custody. Deaths in custody, whether occuring in jail/prison or during an altercation with law enforcement, is a complex issue and requires the forensic pathologist to be knowledgable and deliberative about his/her diagnosis. This paper provides recommendations for the forensic pathologist as it relates to 1) categorization of deaths in custody, 2) critical information required during investigation, 3) enhanced autopsy procedures, 4) guidance on death certification, 5) parameters for statistical reporting, and 6) release of information to the public. A uniform approach by medical examiners and coroners to the investigation and evaluation of deaths in custody is critical. The establishment of recommendations has the potential to ensure consistency and reliability to the definition, investigation, and certification of these cases. Such uniformity and consistency will instill confidence in the independence of the medical examiner/forensic pathologist/coroner by the criminal justice system, public health system, and community at large.