RESUMO
Extracellular vesicles released by tumors (tEVs) disseminate via circulatory networks and promote microenvironmental changes in distant organs favoring metastatic seeding. Despite their abundance in the bloodstream, how hemodynamics affect the function of circulating tEVs remains unsolved. We demonstrated that efficient uptake of tEVs occurs in venous endothelial cells that are subjected to hemodynamics. Low flow regimes observed in veins partially reroute internalized tEVs toward non-acidic and non-degradative Rab14-positive endosomes, at the expense of lysosomes, suggesting that endothelial mechanosensing diverts tEVs from degradation. Subsequently, tEVs promote the expression of pro-angiogenic transcription factors in low flow-stimulated endothelial cells and favor vessel sprouting in zebrafish. Altogether, we demonstrate that low flow regimes potentiate the pro-tumoral function of circulating tEVs by promoting their uptake and rerouting their trafficking. We propose that tEVs contribute to pre-metastatic niche formation by exploiting endothelial mechanosensing in specific vascular regions with permissive hemodynamics.
Assuntos
Vesículas Extracelulares , Neoplasias , Animais , Células Endoteliais , Peixe-Zebra , Vesículas Extracelulares/metabolismo , Hemodinâmica , Neoplasias/patologia , AngiogêneseRESUMO
The vertebrate inner ear employs sensory hair cells and neurons to mediate hearing and balance. In mammals, damaged hair cells and neurons are not regenerated. In contrast, hair cells in the inner ear of zebrafish are produced throughout life and regenerate after trauma. However, it is unknown whether new sensory neurons are also formed in the adult zebrafish statoacoustic ganglion (SAG), the sensory ganglion connecting the inner ear to the brain. Using transgenic lines and marker analysis, we identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In particular, we analyze a Neurod/Nestin-positive progenitor pool that produces large amounts of new neurons at juvenile stages, which transitions to a quiescent state in the adult SAG. Moreover, BrdU pulse chase experiments reveal the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestin-positive progenitor pool at adult stages. Taken together, our study represents the first comprehensive characterization of the adult zebrafish SAG showing that zebrafish, in sharp contrast to mammals, display continued neurogenesis in the SAG well beyond embryonic and larval stages.
Assuntos
Células-Tronco Adultas/fisiologia , Orelha Interna/fisiologia , Gânglios Sensitivos/citologia , Células Ciliadas Auditivas/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Peixe-Zebra , Células-Tronco Adultas/citologia , Envelhecimento/fisiologia , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Orelha Interna/citologia , Embrião não Mamífero , Gânglios Sensitivos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Larva , Proteínas do Tecido Nervoso/metabolismo , Nestina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/fisiologia , Nicho de Células-Tronco/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Among a plethora of functions, extracellular vesicles released by primary tumors spread in the organism and reach distant organs where they can induce the formation of a premetastatic niche. This constitutes a favorable microenvironment for circulating tumor cells which facilitates their seeding and colonization. In this review, we describe the journey of extracellular vesicles (EVs) from the primary tumor to the future metastatic organ, with a focus on the mechanisms used by EVs to target organs with a specific tropism (i.e., organotropism). We then highlight important tumor EV cargos in the context of premetastatic niche formation and summarize their known effects on extracellular matrix remodeling, angiogenesis, vessel permeabilization, resident cell activation, recruitment of foreign cells, and ultimately the formation of a pro-inflammatory and immuno-tolerant microenvironment. Finally, we discuss current experimental limitations and remaining opened questions in light of metastatic diagnosis and potential therapies targeting PMN formation.
RESUMO
The bone marrow microenvironment (BMME) plays a key role in the pathophysiology of myelodysplastic syndromes (MDS), clonal blood disorders affecting the differentiation, and maturation of hematopoietic stem and progenitor cells (HSPCs). In lower-risk MDS patients, ineffective late-stage erythropoiesis can be restored by luspatercept, an activin receptor type IIB ligand trap. Here, we investigated whether luspatercept can modulate the functional properties of mesenchymal stromal cells (MSCs) as key components of the BMME. Luspatercept treatment inhibited Smad2/3 phosphorylation in both healthy and MDS MSCs and reversed disease-associated alterations in SDF-1 secretion. Pre-treatment of MDS MSCs with luspatercept restored the subsequent clonogenic potential of co-cultured HSPCs and increased both their stromal-adherence and their expression of both CXCR4 and ß3 integrin. Luspatercept pre-treatment of MSCs also increased the subsequent homing of co-cultured HSPCs in zebrafish embryos. MSCs derived from patients who had received luspatercept treatment had an increased capacity to maintain the colony forming potential of normal but not MDS HSPCs. These data provide the first evidence that luspatercept impacts the BMME directly, leading to a selective restoration of the ineffective hematopoiesis that is a hallmark of MDS.
Assuntos
Receptores de Activinas Tipo II/farmacologia , Quimiocina CXCL12/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Síndromes Mielodisplásicas/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Adulto , Idoso , Animais , Estudos de Casos e Controles , Quimiocina CXCL12/genética , Hematínicos/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteína Smad2/genética , Proteína Smad2/metabolismo , Células Tumorais Cultivadas , Peixe-ZebraRESUMO
Tumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.
Assuntos
Endotélio Vascular/fisiologia , Hemorreologia , Migração Transendotelial e Transepitelial , Animais , Animais Geneticamente Modificados , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/fisiologia , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Microscopia Intravital , Microfluídica , Microscopia Confocal , Células Neoplásicas Circulantes , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Transdução de Sinais/fisiologia , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra/embriologiaRESUMO
Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.
Assuntos
Neoplasias da Mama/genética , Exossomos/patologia , GTP Fosfo-Hidrolases/metabolismo , Metástase Neoplásica/genética , Animais , Neoplasias da Mama/secundário , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Corpos Multivesiculares/fisiologia , Peixe-ZebraRESUMO
Understanding mechanisms mediating tumor metastasis is crucial for diagnostic and therapeutic targeting. Here, we take advantage of a transparent embryonic zebrafish xenograft model (eZXM) to visualize and track metastatic cells in real time using selective plane illumination microscopy (SPIM) for up to 30 h. Injected human leukemic and breast cancer cells exhibited cell-type specific patterns of intravascular distribution with leukemic cells moving faster than breast cancer cells. Tracking of tumor cells from high-resolution images revealed acute differences in intravascular speed and distance covered by cells. While the majority of injected breast cancer cells predominantly adhered to nearby vasculature, about 30% invaded the non-vascularized tissue, reminiscent of their metastatic phenotype. Survival of the injected tumor cells appeared to be partially inhibited and time-lapse imaging showed a possible role for host macrophages of the recipient embryos. Leukemic cell dissemination could be effectively blocked by pharmacological ROCK1 inhibition using Fasudil. These observations, and the ability to image several embryos simultaneously, support the use of eZXM and SPIM imaging as a functional screening platform to identify compounds that suppress cancer cell spread and invasion.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Neoplasias da Mama/diagnóstico por imagem , Leucemia/diagnóstico por imagem , Metástase Neoplásica/diagnóstico por imagem , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Rastreamento de Células , Feminino , Leucemia/tratamento farmacológico , Microscopia , Invasividade Neoplásica , Metástase Neoplásica/tratamento farmacológico , Transplante de Neoplasias , Imagem com Lapso de Tempo , Peixe-ZebraRESUMO
Hydroxyapatite (HA) nanoparticles are commonly used as building blocks in the design of bone-substituting biomaterials. Recently, these nanoparticles have been considered for the treatment of metastasis disease, since their pH-dependent dissolution behavior allows for precise tuning of release kinetics of loaded cargo. Herein we show that the capacity of drug-loaded nanoparticles stabilized with citrate ions reduce cancer cell survival in an embryonic zebrafish xenograft model. In particular, in vitro studies demonstrate that PtPP-loaded HA nanoparticles exhibit anti-proliferative activity against breast cancer cells at reduced pH. In vivo studies using an embryonic zebrafish xenograft model reveal that PtPP co-delivered with human breast cancer cells strongly reduce cancer cell survival. Similarly, co-injection of breast cancer cells with citrate-functionalized and PtPP-loaded HA nanoparticles into zebrafish significantly reduces survival of cancer cells due to release of chemotherapeutically active kiteplatin species. These results demonstrate the preclinical efficacy of drug-loaded nanoparticles against human breast cancer cells in a xenogenic embryonic in vivo model.
Assuntos
Neoplasias da Mama , Nanopartículas , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Durapatita , Xenoenxertos , Humanos , Platina , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Platinum-based chemotherapeutics exhibit excellent antitumor properties. However, these drugs cause severe side effects including toxicity, drug resistance, and lack of tumor selectivity. Tumor-targeted drug delivery has demonstrated great potential to overcome these drawbacks. Herein, we aimed to design radioactive bisphosphonate-functionalized platinum (195mPt-BP) complexes to confirm preferential accumulation of these Pt-based drugs in metabolically active bone. In vitro NMR studies revealed that release of Pt from Pt BP complexes increased with decreasing pH. Upon systemic administration to mice, Pt-BP exhibited a 4.5-fold higher affinity to bone compared to platinum complexes lacking the bone-seeking bisphosphonate moiety. These Pt-BP complexes formed less Pt-DNA adducts compared to bisphosphonate-free platinum complexes, indicating that in vivo release of Pt from Pt-BP complexes proceeded relatively slow. Subsequently, radioactive 195mPt-BP complexes were synthesized using 195mPt(NO3)2(en) as precursor and injected intravenously into mice. Specific accumulation of 195mPt-BP was observed at skeletal sites with high metabolic activity using micro-SPECT/CT imaging. Furthermore, laser ablation-ICP-MS imaging of proximal tibia sections confirmed that 195mPt BP co-localized with calcium in the trabeculae of mice tibia.