RESUMEN
Calcium phosphosilicate nanoparticles (CPSNPs) are bioresorbable nanoparticles that can be bioconjugated with targeting molecules and encapsulate active agents and deliver them to tumor cells without causing damage to adjacent healthy tissue. Data obtained in this study demonstrated that an anti-CD71 antibody on CPSNPs targets these nanoparticles and enhances their internalization by triple negative breast cancer cells in-vitro. Caspase 3,7 activation, DNA damage, and fluorescent microscopy confirmed the apoptotic breast cancer response caused by targeted anti-CD71-CPSNPs encapsulated with gemcitabine monophosphate, the active metabolite of the chemotherapeutic gemcitabine used to treat cancers including breast and ovarian. Targeted anti-CD71-CPSNPs encapsulated with the fluorophore, Rhodamine WT, were preferentially internalized by breast cancer cells in co-cultures with osteoblasts. While osteoblasts partially internalized anti-CD71-GemMP-CPSNPs, their cell growth was not affected. These results suggest that CPSNPs may be used as imaging tools and selective drug delivery systems for breast cancer that has metastasized to bone.
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Anticuerpos/metabolismo , Compuestos de Calcio/metabolismo , Nanopartículas , Metástasis de la Neoplasia , Osteoblastos/citología , Silicatos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Células 3T3 , Animales , Técnicas de Cocultivo , Femenino , Humanos , Ratones , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Diagnostics based on exosomes and other extracellular vesicles (EVs) are emerging as strategies for informing cancer progression and therapies, since the lipid content and macromolecular cargo of EVs can provide key phenotypic and genotypic information on the parent tumor cell and its microenvironment. We show that EVs derived from more invasive pancreatic tumor cells that express high levels of tumor-specific surface proteins and are composed of highly unsaturated lipids that increase membrane fluidity, exhibit significantly higher conductance versus those derived from less invasive tumor cells, based on dielectrophoresis measurements. Furthermore, through specific binding of the EVs to gold nanoparticle-conjugated antibodies, we show that these conductance differences can be modulated in proportion to the type as well as level of expressed tumor-specific antigens, thereby presenting methods for selective microfluidic enrichment and cytometry-based quantification of EVs based on invasiveness of their parent cell.
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Antígenos de Neoplasias/análisis , Vesículas Extracelulares/química , Proteínas de Neoplasias/análisis , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/patología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Comunicación Celular , Línea Celular Tumoral , Conductividad Eléctrica , Electroforesis , Oro/química , Xenoinjertos , Humanos , Masculino , Nanopartículas del Metal/química , Ratones , Ratones Desnudos , Técnicas Analíticas Microfluídicas , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Páncreas/metabolismo , Páncreas/patología , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/genéticaRESUMEN
OBJECTIVE: To elucidate the genetic architecture of gene expression in pancreatic tissues. DESIGN: We performed expression quantitative trait locus (eQTL) analysis in histologically normal pancreatic tissue samples (n=95) using RNA sequencing and the corresponding 1000 genomes imputed germline genotypes. Data from pancreatic tumour-derived tissue samples (n=115) from The Cancer Genome Atlas were included for comparison. RESULTS: We identified 38 615 cis-eQTLs (in 484 genes) in histologically normal tissues and 39 713 cis-eQTL (in 237 genes) in tumour-derived tissues (false discovery rate <0.1), with the strongest effects seen near transcriptional start sites. Approximately 23% and 42% of genes with significant cis-eQTLs appeared to be specific for tumour-derived and normal-derived tissues, respectively. Significant enrichment of cis-eQTL variants was noted in non-coding regulatory regions, in particular for pancreatic tissues (1.53-fold to 3.12-fold, p≤0.0001), indicating tissue-specific functional relevance. A common pancreatic cancer risk locus on 9q34.2 (rs687289) was associated with ABO expression in histologically normal (p=5.8×10-8) and tumour-derived (p=8.3×10-5) tissues. The high linkage disequilibrium between this variant and the O blood group generating deletion variant in ABO (exon 6) suggested that nonsense-mediated decay (NMD) of the 'O' mRNA might explain this finding. However, knockdown of crucial NMD regulators did not influence decay of the ABO 'O' mRNA, indicating that a gene regulatory element influenced by pancreatic cancer risk alleles may underlie the eQTL. CONCLUSIONS: We have identified cis-eQTLs representing potential functional regulatory variants in the pancreas and generated a rich data set for further studies on gene expression and its regulation in pancreatic tissues.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Expresión Génica , Páncreas , Neoplasias Pancreáticas/genética , Sitios de Carácter Cuantitativo , ARN Neoplásico/análisis , Transcriptoma , Alelos , Cromosomas Humanos Par 9 , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Degradación de ARNm Mediada por Codón sin Sentido , Polimorfismo de Nucleótido Simple , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ARNRESUMEN
Genome-wide association studies (GWAS) have identified multiple common susceptibility loci for pancreatic cancer. Here we report fine-mapping and functional analysis of one such locus residing in a 610 kb gene desert on chr13q22.1 (marked by rs9543325). The closest candidate genes, KLF5, KLF12, PIBF1, DIS3 and BORA, range in distance from 265-586 kb. Sequencing three sub-regions containing the top ranked SNPs by imputation P-value revealed a 30 bp insertion/deletion (indel) variant that was significantly associated with pancreatic cancer risk (rs386772267, P = 2.30 × 10-11, OR = 1.22, 95% CI 1.15-1.28) and highly correlated to rs9543325 (r2 = 0.97 in the 1000 Genomes EUR population). This indel was the most significant cis-eQTL variant in a set of 222 histologically normal pancreatic tissue samples (ß = 0.26, P = 0.004), with the insertion (risk-increasing) allele associated with reduced DIS3 expression. DIS3 encodes a catalytic subunit of the nuclear RNA exosome complex that mediates RNA processing and decay, and is mutated in several cancers. Chromosome conformation capture revealed a long range (570 kb) physical interaction between a sub-region of the risk locus, containing rs386772267, and a region â¼6 kb upstream of DIS3 Finally, repressor regulatory activity and allele-specific protein binding by transcription factors of the TCF/LEF family were observed for the risk-increasing allele of rs386772267, indicating that expression regulation at this risk locus may be influenced by the Wnt signaling pathway. In conclusion, we have identified a putative functional indel variant at chr13q22.1 that associates with decreased DIS3 expression in carriers of pancreatic cancer risk-increasing alleles, and could therefore affect nuclear RNA processing and/or decay.
Asunto(s)
Cromosomas Humanos Par 13 , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Neoplasias Pancreáticas/genética , Alelos , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Mapeo Cromosómico/métodos , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Sitios Genéticos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Mutación INDEL , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/genéticaRESUMEN
Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive, phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained biological activity. Analysis of orthotopic tumors from mice treated systemically with tumor-targeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells and release of active drug cargos intracellularly. These findings demonstrate a novel methodology to efficiently encapsulate chemotherapeutic agents into the CPSNPs and to effectively deliver them to pancreatic tumor cells.
Asunto(s)
Antineoplásicos/administración & dosificación , Compuestos de Calcio/química , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Fluorouracilo/administración & dosificación , Nanopartículas/química , Neoplasias Pancreáticas/tratamiento farmacológico , Silicatos/química , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/química , Desoxicitidina/uso terapéutico , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Fluorouracilo/análogos & derivados , Fluorouracilo/uso terapéutico , Humanos , Masculino , Ratones , Ratones Desnudos , Nanopartículas/ultraestructura , Fosforilación , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
BACKGROUND: Obesity and dietary fat are associated with increased risk of several malignancies including pancreatic cancer. The incidence of pancreatic cancer is increased in countries that consume diets high in fat. AIM: The purpose of this study was to assess the relationship and mechanism of action between dietary fat and endogenous cholecystokinin (CCK) on pancreatic tumor growth and metastasis in an immunocompetent animal model. METHODS: C57BL/6 mice were placed on regular, low-fat, or high-fat diets for 8 weeks before establishment of Panc-02 orthotopic pancreatic tumors. Mice were then treated with a CCK-A receptor antagonist, devazepide, or vehicle for an additional 2.5 weeks. Pancreas tumors were weighed and metastases counted. Blood CCK levels were measured by radioimmunoassay (RIA). Tissues were examined histologically and studied for genes associated with metastasis by RT-PCR array. Effects of the CCK antagonist on Panc-02 cells invasiveness was assessed in a Matrigel invasion assay. RESULTS: Mice that received the high-fat diet had larger tumors and tenfold higher serum CCK levels by RIA compared to normal diet controls (p < 0.01). Pancreatic tumors in high-fat diet mice treated with the antagonist had fewer intravascular tumor emboli and metastases compared to controls. The reduction in tumor emboli correlated with decreased vascular endothelial growth factor-A (VEGF-A) expression in tumors (p < 6 × 10(-9)). In vitro invasiveness of Panc-02 cells also was reduced by CCK-A receptor antagonist treatment (p = 1.33 × 10(-6)). CONCLUSION: CCK is a mediator of dietary fat-associated pancreatic cancer. CCK is also involved in the invasiveness of pancreatic tumors through a mechanism involving VEGF-A.
Asunto(s)
Colecistoquinina/metabolismo , Grasas de la Dieta/efectos adversos , Neoplasias Pancreáticas/metabolismo , Animales , Glucemia , Línea Celular Tumoral , Devazepida/farmacología , Grasas de la Dieta/administración & dosificación , Relación Dosis-Respuesta a Droga , Embolia/prevención & control , Antagonistas de Hormonas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Metástasis de la Neoplasia/prevención & control , Neoplasias Pancreáticas/patología , RadioinmunoensayoRESUMEN
Vascular cell adhesion molecule-1 (VCAM-1) was identified over 2 decades ago as an endothelial adhesion receptor involved in leukocyte recruitment and cell-based immune responses. In atherosclerosis, a chronic inflammatory disease of the blood vessels that is the leading cause of death in the USA, endothelial VCAM-1 is robustly expressed beginning in the early stages of the disease. The interactions of circulating immune cells with VCAM-1 on the activated endothelial cell surface promote the uptake of monocytes and the progression of atherosclerotic lesions in susceptible vessels. Herein, we review the role of VCAM-1 in atherosclerosis and the use of VCAM-1 binding peptides, antibodies and aptamers as targeting agents for nanoplatforms for early detection and treatment of atherosclerotic disease.
Asunto(s)
Aterosclerosis , Nanopartículas , Humanos , Molécula 1 de Adhesión Celular Vascular/metabolismo , Aterosclerosis/diagnóstico , Aterosclerosis/tratamiento farmacológico , Péptidos/metabolismo , Membrana Celular/metabolismo , Nanopartículas/uso terapéutico , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Adhesión CelularRESUMEN
The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.
RESUMEN
Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly(100) and Ser(101) on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation.
Asunto(s)
Metaloendopeptidasas/metabolismo , Monocitos/fisiología , Ocludina/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Perros , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Humanos , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Permeabilidad/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
Gastrin stimulates the growth of pancreatic cancer cells through the activation of the cholecystokinin-B receptor (CCK-BR), which has been found to be overexpressed in pancreatic cancer. In this study, we proposed that the CCK-BR drives growth of pancreatic cancer; hence, interruption of CCK-BR activity could potentially be an ideal target for cancer therapeutics. The effect of CCK-BR downregulation in the human pancreatic adenocarcinoma cells was examined by utilizing specific CCK-BR-targeted RNA interference reagents. The CCK-BR receptor expression was both transiently and stably downregulated by transfection with selective CCK-BR small-interfering RNA or short-hairpin RNA, respectively, and the effects on cell growth and apoptosis were assessed. CCK-BR downregulation resulted in reduced cancer cell proliferation, decreased DNA synthesis, and cell cycle arrest as demonstrated by an inhibition of G(1) to S phase progression. Furthermore, CCK-BR downregulation increased caspase-3 activity, TUNEL-positive cells, and decreased X-linked inhibitor of apoptosis protein expression, suggesting apoptotic activity. Pancreatic cancer cell mobility was decreased when the CCK-BR was downregulated, as assessed by a migration assay. These results show the importance of the CCK-BR in regulation of growth and apoptosis in pancreatic cancer. Strategies to decrease the CCK-BR expression and activity may be beneficial for the development of new methods to improve the treatment for patients with pancreatic cancer.
Asunto(s)
Apoptosis/genética , Proliferación Celular , Regulación hacia Abajo , Páncreas/metabolismo , Receptor de Colecistoquinina B/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Interferencia de ARN , Receptor de Colecistoquinina B/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
MEP1A, which encodes the α subunit of meprin metalloproteinases, is a susceptibility gene for inflammatory bowel disease (IBD), and decreased intestinal meprin-α expression is associated with enhanced IBD in humans. Mice lacking meprin α (α knockout, αKO) have more severe colitis induced by dextran sulfate sodium (DSS) than wild-type (WT) mice, indicating an anti-inflammatory role for meprin A. Previous studies and those herein indicate the meprin B has proinflammatory activities. Therefore, mice lacking both meprin A and B (dKO mice) were generated to determine how their combined absence alters the inflammatory response to DSS. Unchallenged dKO mice grow and reproduce normally and have no obvious abnormal phenotype, except for a slightly elevated plasma albumin in both males and females and a lower urine creatinine level in dKO males. Upon oral administration of 3.5% DSS, the dKO mice have more severe colitis than the WT and ßKO mice but significantly less than the αKO mice. The dKO mice lose more weight and have elevated MPO and IL-6 activities in the colon compared with WT mice. Systemic inflammation, monitored by plasma nitric oxide levels, is absent in DSS-treated dKO mice, unlike WT mice. The severity of experimental IBD in dKO mice is intermediate between αKO and WT mice. The data indicate that the absence of meprin A aggravates chronic inflammation and the lack of meprin B affords some protection from injury. Manipulation of the expression of meprin gene products may have therapeutic potential.
Asunto(s)
Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Metaloendopeptidasas/metabolismo , Administración Oral , Animales , Antiinflamatorios/metabolismo , Enfermedad Crónica , Colitis/metabolismo , Colitis/patología , Sulfato de Dextran/administración & dosificación , Progresión de la Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/genética , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Metaloendopeptidasas/deficiencia , Ratones , Ratones Noqueados , Permeabilidad , Peroxidasa/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
PURPOSE: Accurate tumor identification and staging can be difficult. Aptamer-targeted indocyanine green (ICG)-nanoparticles can enhance near-infrared fluorescent imaging of pancreatic and prostate tumors and could improve early cancer detection. This project explored whether calcium-phosphosilicate nanoparticles, also known as NanoJackets (NJs), that were bioconjugated with a tumor-specific targeting DNA aptamer could improve the non-invasive detection of pancreatic and prostate tumors. METHODS: Using in vivo near-infrared optical imaging and ex vivo fluorescence analysis, DNA aptamer-targeted ICG-loaded NJs were compared to untargeted NJs for detection of tumors. RESULTS: Nanoparticles were bioconjugated with the DNA aptamer AP1153, which binds to the CCK-B receptor (CCKBR). Aptamer bioconjugated NJs were not significantly increased in size compared with unconjugated nanoparticles. AP1153-ICG-NJ accumulation in orthotopic pancreatic tumors peaked at 18 h post-injection and the ICG signal was cleared by 36 h with no evidence on uptake by non-tumor tissues. Ex vivo tumor imaging confirmed the aptamer-targeted NJs accumulated to higher levels than untargeted NJs, were not taken up by normal pancreas, exited from the tumor vasculature, and were well-dispersed throughout pancreatic and prostate tumors despite extensive fibrosis. Specificity for AP1153-NJ binding to the CCK-B receptor on pancreatic tumor cells was confirmed by pre-treating tumor-bearing mice with the CCK receptor antagonist proglumide. Proglumide pre-treatment reduced the in vivo tumoral accumulation of AP1153-NJs to levels comparable to that of untargeted NJs. CONCLUSION: Through specific interactions with CCK-B receptors, tumor-targeted nanoparticles containing either ICG or rhodamine WT were well distributed throughout the matrix of both pancreatic and prostate tumors. Tumor-targeted NJs carrying various imaging agents can enhance tumor detection.
Asunto(s)
Aptámeros de Nucleótidos/química , Diagnóstico por Imagen , Nanopartículas/química , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias de la Próstata/diagnóstico por imagen , Silicatos/química , Animales , Calcio , Línea Celular Tumoral , Colorantes , Fluorescencia , Humanos , Verde de Indocianina/química , Rayos Infrarrojos , Masculino , Ratones , Neovascularización Patológica/diagnóstico por imagen , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias de la Próstata/irrigación sanguínea , Receptores de Colecistoquinina/metabolismo , Rodaminas/química , Microambiente TumoralRESUMEN
It is estimated that early detection of pancreatic ductal adenocarcinoma (PDAC) could increase long-term patient survival by as much as 30% to 40% (Seufferlein, T. et al., Nat. Rev. Gastroenterol. Hepatol.2016, 13, 74â»75). There is an unmet need for reagents that can reliably identify early cancerous or precancerous lesions through various imaging modalities or could be employed to deliver anticancer treatments specifically to tumor cells. However, to date, many PDAC tumor-targeting strategies lack selectivity and are unable to discriminate between tumor and nontumor cells, causing off-target effects or unclear diagnoses. Although a variety of approaches have been taken to identify tumor-targeting reagents that can effectively direct therapeutics or imaging agents to cancer cells (Liu, D. et al., J. Controlled Release2015, 219, 632â»643), translating these reagents into clinical practice has been limited, and it remains an area open to new methodologies and reagents (O'Connor, J.P. et al., Nat. Rev. Clin. Oncol. 2017, 14, 169â»186). G proteinâ»coupled receptors (GPCRs), which are key target proteins for drug discovery and comprise a large proportion of currently marketed therapeutics, hold significant promise for tumor imaging and targeted treatment, particularly for pancreatic cancer.
RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages (TAMs), yet the mechanisms by which tumor cells and TAMs communicate are not fully understood. Here we show that exosomes secreted by PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages in vitro. Exosomes from AsPC-1, an ascites-derived human PDAC cell line, were enriched in ICAM-1, which mediated their docking to macrophages through interactions with surface-exposed CD11c on macrophages. AsPC-1 exosomes also contained much higher levels of arachidonic acid (AA), and they fused at a higher rate with THP-1-derived macrophages than did exosomes from other PDAC cell lines or from an immortalized normal pancreatic ductal epithelial cell line (HPDE) H6c7. Phospholipase A2 enzymatic cleavage of arachidonic acid from AsPC-1 exosomes reduced fusion efficiency. PGE2 secretion was elevated in macrophages treated with AsPC-1 exosomes but not in macrophages treated with exosomes from other cell lines, suggesting a functional role for the AsPC-1 exosome-delivered arachidonic acid in macrophages. Non-polarized (M0) macrophages treated with AsPC-1 exosomes had increased levels of surface markers indicative of polarization to an immunosuppressive M2-like phenotype (CD14hi CD163hi CD206hi). Furthermore, macrophages treated with AsPC-1 exosomes had significantly increased secretion of pro-tumoral, bioactive molecules including VEGF, MCP-1, IL-6, IL-1ß, MMP-9, and TNFα. Together, these results demonstrate that compared to exosomes from other primary tumor-derived PDAC cell lines, AsPC-1 exosomes alter THP-1-derived macrophage phenotype and function. AsPC-1 exosomes mediate communication between tumor cells and TAMs that contributes to tumor progression.
Asunto(s)
Exosomas , Macrófagos/metabolismo , Neoplasias Pancreáticas/metabolismo , Ácido Araquidónico/metabolismo , Línea Celular Tumoral , Humanos , Terapia de Inmunosupresión , Neoplasias PancreáticasRESUMEN
Breast cancer is a major ongoing public health issue among women in both developing and developed countries. Significant progress has been made to improve the breast cancer treatment in the past decades. However, the current clinical approaches are invasive, of low specificity and can generate severe side effects. As a rapidly developing field, nanotechnology brings promising opportunities to human cancer diagnosis and treatment. The use of nanoparticulate-based platforms overcomes biological barriers and allows prolonged blood circulation time, simultaneous tumor targeting and enhanced accumulation of drugs in tumors. Currently available and clinically applicable innovative nanoparticulate-based systems for breast cancer nanotherapies are discussed in this review.
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Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Animales , Transporte Biológico , Liberación de Fármacos , Femenino , Humanos , Nanomedicina/métodos , Tamaño de la Partícula , Permeabilidad , Propiedades de SuperficieRESUMEN
Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against "exposed" CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bioconjugation of AP1153 to the surface of fluorescent NPs greatly facilitated delivery of NPs to PDAC tumors in vivo. The selectivity of this AP-targeted NP delivery system holds promise for enhanced early detection of PDAC lesions as well as improved chemotherapeutic treatments for PDAC patients.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Carcinoma Ductal Pancreático/terapia , Nanoconjugados/administración & dosificación , Neoplasias Pancreáticas/terapia , Receptor de Colecistoquinina B/uso terapéutico , Animales , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Células COS , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Sistemas de Liberación de Medicamentos , Humanos , Imagenología Tridimensional , Masculino , Ratones , Ratones Desnudos , Microscopía Confocal , Nanoconjugados/química , Imagen Óptica , Neoplasias Pancreáticas/metabolismo , Receptor de Colecistoquinina B/genética , Receptor de Colecistoquinina B/metabolismo , Nanomedicina Teranóstica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Here we describe isolation and characterization of macrophage-tumor cell fusions (MTFs) from the blood of pancreatic ductal adenocarcinoma (PDAC) patients. The MTFs were generally aneuploidy, and immunophenotypic characterizations showed that the MTFs express markers characteristic of PDAC and stem cells, as well as M2-polarized macrophages. Single cell RNASeq analyses showed that the MTFs express many transcripts implicated in cancer progression, LINE1 retrotransposons, and very high levels of several long non-coding transcripts involved in metastasis (such as MALAT1). When cultured MTFs were transplanted orthotopically into mouse pancreas, they grew as obvious well-differentiated islands of cells, but they also disseminated widely throughout multiple tissues in "stealth" fashion. They were found distributed throughout multiple organs at 4, 8, or 12 weeks after transplantation (including liver, spleen, lung), occurring as single cells or small groups of cells, without formation of obvious tumors or any apparent progression over the 4 to 12 week period. We suggest that MTFs form continually during PDAC development, and that they disseminate early in cancer progression, forming "niches" at distant sites for subsequent colonization by metastasis-initiating cells.
Asunto(s)
Carcinoma Ductal Pancreático/sangre , Carcinoma Ductal Pancreático/patología , Macrófagos/patología , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/ultraestructura , Fusión Celular , Núcleo Celular/patología , Humanos , Imagenología Tridimensional , Inmunohistoquímica , Inmunofenotipificación , Masculino , Ratones Desnudos , Microscopía Confocal , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/ultraestructura , Ploidias , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
OBJECTIVES: Today, genetic biomarkers have been demonstrated to play an important role in identifying at-risk subjects for familial or inherited cancers. We have identified a single-nucleotide polymorphism (SNP) that results in missplicing of the cholecystokinin (CCK) receptor gene and expressing a larger mutated receptor in pancreatic cancer. The purpose of this study was to evaluate the significance and specificity of this SNP as a potential biomarker in patients with pancreatic cancer compared with other gastrointestinal (GI) cancers that also have CCK receptors. METHODS: DNA was isolated and genotyped for the CCK receptor SNP from frozen tumor tissue from banked specimens of patients with pancreas, gastric, or colon cancer and from human cancer cell lines. Genotype and allelic frequencies were compared between the cancer cohort and two normal control databases using Fisher's exact test and odds ratio (OR). The Kaplan-Meier method was used to estimate the survival for patients with the CCK-B receptor SNP compared with those with the wild-type genotype. Immunohistochemical staining of cancer cells was done to detect the mutated receptor. RESULTS: Colon and gastric cancer patients had similar genotype frequencies for the CCK receptor SNP as that reported in the normal population. In contrast, the prevalence of the SNP in subjects with pancreatic cancer was twice that of controls and other GI cancers. Survival was adversely affected by the presence of the SNP only in those with pancreatic cancer. Immunoreactivity for the mutated receptor was positive in pancreatic cancer tissues with the SNP but absent in other GI cancers. CONCLUSIONS: A SNP of the CCK receptor is significantly increased in patients with pancreatic cancer but not in those with other GI malignancies. Therefore, this SNP may be a potential biomarker for pancreatic cancer.
RESUMEN
Meprins are unique plasma membrane and secreted metalloproteinases that are highly regulated at the transcriptional and post-translational levels. Meprin alpha and beta subunits are abundantly expressed in kidney and intestinal epithelial cells, are secreted into the urinary tract and intestinal lumen, and are found in leukocytes and cancer cells under certain conditions. Their location and proteolytic activities indicate functions at the interface of the host and the external environment, and in trafficking of macrophages and metastases of cancer cells. These proteases can be detrimental when there is tissue damage or disruption, as in acute renal injury or intestinal inflammation, and there is evidence they are involved in movement of leukocytes and cancer cells to sites of infection or in metastasis, respectively.
Asunto(s)
Enfermedades Gastrointestinales/enzimología , Regulación Enzimológica de la Expresión Génica , Enfermedades Renales/enzimología , Metaloendopeptidasas/metabolismo , Neoplasias/enzimología , Infecciones Urinarias/enzimología , Animales , Humanos , Metaloendopeptidasas/genética , Neoplasias/genética , Infecciones Urinarias/genéticaRESUMEN
Inhibition of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by the irreversible inhibitor alpha-difluoromethylornithine (DFMO) has been shown to decrease the invasiveness of metastatic human breast cancer cell lines. However, the mechanism by which DFMO acts to reduce invasiveness is unclear. Using the human breast cancer cell line MDA-MB-435, the effect of DFMO on metalloprotease gene expression was investigated. DFMO treatment decreases the expression of the metalloprotease meprin alpha, while concurrent treatment with DFMO and the polyamine putrescine partially restored meprin alpha expression levels. Expression of MMP-7 mRNA was reduced by DFMO, while MMPs-1, -2, -3, -14, and meprin beta were unaffected. Treatment of cells with a second inhibitor of polyamine biosynthesis, the S-adenosylmethionine decarboxylase (SAMDC) inhibitor SAM486A, also resulted in a dosage dependent decrease in meprin alpha and MMP-7 mRNA. In addition, DFMO treatment decreased meprin alpha at the protein level by 2 days of treatment, and MMP-7 protein levels at 4 and 6 days. Previous studies have shown that DFMO treatment increases ERK phosphorylation and signaling through the MAP kinase pathway. The decrease in meprin alpha expression was reversed with the MEK inhibitor PD98059, demonstrating that MAP kinase signaling mediates the effect of DFMO and SAM486A. MDA-MB-435 cells treated with the meprin alpha inhibitor actinonin (5 nM) were less invasive in vitro, indicating that meprin alpha is mechanistically involved in invasion. The decrease in meprin alpha expression in DFMO and SAM486A-treated cells indicates a means by which these compounds can decrease the invasiveness of metastatic breast cancer cells.