RESUMEN
Most patients diagnosed with resected pancreatic adenocarcinoma (PDAC) survive less than 5 years, but a minor subset survives longer. Here, we dissect the role of the tumor microbiota and the immune system in influencing long-term survival. Using 16S rRNA gene sequencing, we analyzed the tumor microbiome composition in PDAC patients with short-term survival (STS) and long-term survival (LTS). We found higher alpha-diversity in the tumor microbiome of LTS patients and identified an intra-tumoral microbiome signature (Pseudoxanthomonas-Streptomyces-Saccharopolyspora-Bacillus clausii) highly predictive of long-term survivorship in both discovery and validation cohorts. Through human-into-mice fecal microbiota transplantation (FMT) experiments from STS, LTS, or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration. Our study demonstrates that PDAC microbiome composition, which cross-talks to the gut microbiome, influences the host immune response and natural history of the disease.
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Carcinoma Ductal Pancreático/microbiología , Carcinoma Ductal Pancreático/mortalidad , Microbioma Gastrointestinal , Neoplasias Pancreáticas/microbiología , Neoplasias Pancreáticas/mortalidad , Adulto , Anciano , Animales , Bacterias/clasificación , Línea Celular Tumoral , Estudios de Cohortes , Trasplante de Microbiota Fecal , Heces/microbiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , ARN Ribosómico 16S/genética , Análisis de Secuencia de ARN , Tasa de SupervivenciaRESUMEN
BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.
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Aterosclerosis , Placa Aterosclerótica , Animales , Ratones , Aterosclerosis/metabolismo , Inflamación , Proteína Quinasa 7 Activada por Mitógenos/genética , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
Oxidative stress (OS) induced activation of p38 mitogen-activated kinase (MAPK) and cell fate from p38 signaling was tested using the human fetal membrane's amnion epithelial cells (AEC). We created p38 KO AEC using the CRISPR/Cas9 approach and tested cell fate in response to OS on an AEC-free fetal membrane extracellular matrix (ECM). Screening using image CyTOF indicated OS causing epithelial-mesenchymal transition (EMT). Further testing revealed p38 deficiency prevented AEC senescence, EMT, cell migration, and inflammation. To functionally validate in vitro findings, fetal membrane-specific conditional KO (cKO) mice were developed by injecting Cre-recombinase encoded exosomes intra-amniotically into p38αloxP/loxP mice. Amnion membranes from p38 cKO mice had reduced senescence, EMT, and increased anti-inflammatory IL-10 compared with WT animals. Our study suggested that overwhelming activation of p38 in response to OS inducing risk exposures can have an adverse impact on cells, cause cell invasion, inflammation, and ECM degradation detrimental to tissue homeostasis.
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Mitógenos , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , Ratones , Animales , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Células Epiteliales/metabolismo , Amnios , Inflamación/metabolismoRESUMEN
BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.
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Inestabilidad Cromosómica , Neoplasias Colorrectales/genética , Neoplasias Experimentales/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Aurora Quinasa B/metabolismo , Azoximetano/toxicidad , Carcinogénesis/genética , Línea Celular Tumoral , Colon/citología , Colon/patología , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/patología , Análisis Citogenético , Dextranos/toxicidad , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/patología , Organoides , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genéticaRESUMEN
Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity,is frequently altered in human cancers including mantle cell lymphoma (MCL). Loss of ATM protein is linked to accumulation of nonfunctional mitochondria and defective mitophagy, in both murine thymocytes and in A-T cells. However, the mechanistic role of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that FCCP-induced mitophagy in MCL and other cancer cell lines is dependent on ATM but independent of its kinase function. While Granta-519 MCL cells possess single copy and kinase dead ATM and are resistant to FCCP-induced mitophagy, both Jeko-1 and Mino cells are ATM proficient and induce mitophagy. Stable knockdown of ATM in Jeko-1 and Mino cells conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mROS. ATM interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner. Knockdown of ATM in HeLa cells resulted in proteasomal degradation of GFP-Parkin which was rescued by the proteasome inhibitor, MG132 suggesting that ATM-Parkin interaction is important for Parkin stability. Neither loss of ATM kinase activity in primary B cell lymphomas nor inhibition of ATM kinase in MCL, A-T and HeLa cell lines mitigated FCCP or CCCP-induced mitophagy suggesting that ATM kinase activity is dispensable for mitophagy. Malignant B-cell lymphomas without detectable ATM, Parkin, Pink1, and Parkin-Ub ser65 phosphorylation were resistant to mitophagy, providing the first molecular evidence of ATM's role in mitophagy in MCL and other B-cell lymphomas.
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Proteínas de la Ataxia Telangiectasia Mutada , Ataxia Telangiectasia , Linfoma de Células del Manto , Adulto , Animales , Células HeLa , Humanos , Linfoma de Células del Manto/genética , Ratones , Mitofagia/genética , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. In vivo, the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML.
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Leucemia Mieloide Aguda , Proteómica , Apoptosis , Azetidinas , Compuestos Bicíclicos Heterocíclicos con Puentes , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Piperidinas , Proteínas Proto-Oncogénicas c-bcl-2/genética , SulfonamidasRESUMEN
BACKGROUND: Single rare cell characterization represents a new scientific front in personalized therapy. Imaging mass cytometry (IMC) may be able to address all these questions by combining the power of MS-CyTOF and microscopy. METHODS: We have investigated this IMC method using < 100 to up to 1000 cells from human sarcoma tumor cell lines by incorporating bioinformatics-based t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis of highly multiplexed IMC imaging data. We tested this process on osteosarcoma cell lines TC71, OHS as well as osteosarcoma patient-derived xenograft (PDX) cell lines M31, M36, and M60. We also validated our analysis using sarcoma patient-derived CTCs. RESULTS: We successfully identified heterogeneity within individual tumor cell lines, the same PDX cells, and the CTCs from the same patient by detecting multiple protein targets and protein localization. Overall, these data reveal that our t-SNE-based approach can not only identify rare cells within the same cell line or cell population, but also discriminate amongst varied groups to detect similarities and differences. CONCLUSIONS: This method helps us make greater inroads towards generating patient-specific CTC fingerprinting that could provide an accurate tumor status from a minimally-invasive liquid biopsy.
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Neoplasias Óseas/patología , Citometría de Imagen/métodos , Células Neoplásicas Circulantes/patología , Osteosarcoma/patología , Análisis por Matrices de Proteínas/métodos , Actinas/análisis , Biopsia con Aguja Fina , Línea Celular Tumoral , Biología Computacional , Variaciones en el Número de Copia de ADN , Dermatoglifia del ADN , Humanos , Biopsia Líquida , Vimentina/análisisRESUMEN
In acute myeloid leukemia (AML), high Galectin 3 (LGALS3) expression is associated with poor prognosis. The role of LGALS3 derived from mesenchymal stromal cells (MSC) in the AML microenvironment is unclear; however, we have recently found high LGALS3 expression in MSC derived from AML patients is associated with relapse. In this study, we used reverse phase protein analysis (RPPA) to correlate LGALS3 expression in AML MSC with 119 other proteins including variants of these proteins such as phosphorylated forms or cleaved forms to identify biologically relevant pathways. RPPA revealed that LGALS3 protein was positively correlated with expression of thirteen proteins including MYC, phosphorylated beta-Catenin (p-CTNNB1), and AKT2 and negatively correlated with expression of six proteins including integrin beta 3 (ITGB3). String analysis revealed that proteins positively correlated with LGALS3 showed strong interconnectivity. Consistent with the RPPA results, LGALS3 suppression by shRNA in MSC resulted in decreased MYC and AKT expression while ITGB3 was induced. In co-culture, the ability of AML cell to adhere to MSC LGALS3 shRNA transductants was reduced compared to AML cell adhesion to MSC control shRNA transductants. Finally, use of novel specific LGALS3 inhibitor CBP.001 in co-culture of AML cells with MSC reduced viable leukemia cell populations with induced apoptosis and augmented the chemotherapeutic effect of AraC. In summary, the current study demonstrates that MSC-derived LGALS3 may be critical for important biological pathways for MSC homeostasis and for regulating AML cell localization and survival in the leukemia microenvironmental niche.
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Galectina 3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células Madre Mesenquimatosas/metabolismo , Regulación hacia Arriba , Proteínas Sanguíneas , Técnicas de Cocultivo , Galectinas , Regulación Neoplásica de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Fosforilación , Mapas de Interacción de Proteínas , Proteómica , Células THP-1 , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
INTRODUCTION: We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. MATERIALS AND METHODS: Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44+CD49f+CD133/2+ mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. RESULTS: 8 of 8 IBC samples expressed isolated CD44+CD49f+CD133/2+ stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44+CD49f+CD133/2+ cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). CONCLUSIONS: Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.
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Neoplasias Inflamatorias de la Mama/inmunología , Neoplasias Inflamatorias de la Mama/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Células Madre/metabolismo , Biomarcadores , Línea Celular Tumoral , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Inflamatorias de la Mama/genética , Neoplasias Inflamatorias de la Mama/patología , Macrófagos/patología , Clasificación del Tumor , Estadificación de Neoplasias , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: Oesophageal squamous cell carcinoma (ESCC) is a heterogeneous disease with variable outcomes that are challenging to predict. A better understanding of the biology of ESCC recurrence is needed to improve patient care. Our goal was to identify small non-coding RNAs (sncRNAs) that could predict the likelihood of recurrence after surgical resection and to uncover potential molecular mechanisms that dictate clinical heterogeneity. DESIGN: We developed a robust prediction model for recurrence based on the analysis of the expression profile data of sncRNAs from 108 fresh frozen ESCC specimens as a discovery set and assessment of the associations between sncRNAs and recurrence-free survival (RFS). We also evaluated the mechanistic and therapeutic implications of sncRNA obtained through integrated analysis from multiple datasets. RESULTS: We developed a risk assessment score (RAS) for recurrence with three sncRNAs (microRNA (miR)-223, miR-1269a and nc886) whose expression was significantly associated with RFS in the discovery cohort (n=108). RAS was validated in an independent cohort of 512 patients. In multivariable analysis, RAS was an independent predictor of recurrence (HR, 2.27; 95% CI, 1.26 to 4.09; p=0.007). This signature implies the expression of ΔNp63 and multiple alterations of driver genes like PIK3CA. We suggested therapeutic potentials of immune checkpoint inhibitors in low-risk patients, and Polo-like kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and histone deacetylase inhibitors in high-risk patients. CONCLUSION: We developed an easy-to-use prognostic model with three sncRNAs as robust prognostic markers for postoperative recurrence of ESCC. We anticipate that such a stratified and systematic, tumour-specific biological approach will potentially contribute to significant improvement in ESCC treatment.
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Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , MicroARNs/análisis , Recurrencia Local de Neoplasia/genética , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/cirugía , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Supervivencia sin Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/cirugía , Femenino , Genómica , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Modelos Biológicos , Terapia Molecular Dirigida , Fosfatidilinositol 3-Quinasas/genética , Valor Predictivo de las Pruebas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Medición de Riesgo , Biología de Sistemas , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Quinasa Tipo Polo 1RESUMEN
Galectin 3 (LGALS3) expression is prognostic for poor survival in acute myeloid leukemia (AML) patients. GCS-100 is a novel galectin inhibitor that may prove useful for AML therapy. In this study, we found that GCS-100 induced apoptosis in AML cells. The agent reduced MCL-1 expression suggesting that GCS-100 could be more effective when combined with a BH3 mimetic. Indeed, potent synergistic cytotoxicity was achieved when GCS-100 was combined with ABT-737 or ABT-199. Furthermore, the GCS-100/ABT-199 combination was effective against primary AML blast cells from patients with FLT3 ITD mutations, which is another prognostic factor for poor outcome in AML. This activity may involve wild-type p53 as shRNA knockdown of LGALS3 or galectin 1 (LGALS1) sensitized wild-type p53 OCI-AML3 cells to GCS-100/ABT-737-induced apoptosis to a much greater extent than p53 null THP-1 cells. Suppression of LGALS3 by shRNA inhibited MCL-1 expression in OCI-AML3 cells, but not THP-1 cells, suggesting the induced sensitivity to ABT-737 may involve a MCL-1 mediated mechanism. OCI-AML3 cells with LGALS1 shRNA were also sensitized to ABT-737. However, these cells exhibited increased MCL-1 expression, so MCL-1 reduction is apparently not required in this process. A role for p53 appears important as GCS-100 induces p53 expression and shRNA knockdown of p53 protected OCI-AML3 cells from the cytotoxic effects of the GCS-100/ABT-737 treatment combination. Our results suggest that galectins regulate a survival axis in AML cells, which may be targeted via combined inhibition with drugs such as GCS-100 and ABT-199.
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Compuestos de Bifenilo/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Leucemia Mieloide Aguda/patología , Nitrofenoles/farmacología , Polisacáridos/farmacología , Sulfonamidas/farmacología , Proteína p53 Supresora de Tumor/genética , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos de Bifenilo/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Galectinas/antagonistas & inhibidores , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Nitrofenoles/administración & dosificación , Fragmentos de Péptidos/química , Piperazinas/administración & dosificación , Piperazinas/farmacología , Polisacáridos/administración & dosificación , Proteínas Proto-Oncogénicas/química , Sulfonamidas/administración & dosificaciónRESUMEN
OBJECTIVE: To aim of this study was to determine the clinical and biological prognostic factors for locoregional recurrence (LRR) in patients with thoracic esophageal squamous cell carcinoma (ESCC) undergoing radical two-field lymph node dissection (2FLD). METHODS: A total of 462 patients diagnosed with thoracic ESCC underwent radical esophagectomy between March 2001 and May 2010 at Sun Yat-Sen University Cancer Center. Clinical characteristics, CD44 expression, and tumor-infiltrating lymphocyte (TIL) levels were evaluated in 198 patients who underwent R0 dissection with long-term follow-up. Partial Cox regression analysis with leave-one-out cross-validation was performed to validate the selected risk factors. RESULTS: With a median follow-up of 54 months, the 5-year local failure-free survival (LFFS) rate of 198 patients was 62.5%. Multivariate analysis revealed that T stage (p = 0.043), pathological positive tumor above the carina (p = 0.000), CD44 expression level (p = 0.045) and TIL level (p = 0.007) were prognostic factors for LFFS, while the Cox model with risk scores had an area under the curve value of 83.6% for the prediction of 5-year LFFS. The best cut-off value (sum score = 11.19) was used to determine the high- and low-risk groups, with patients at high risk having a significantly shorter 5-year LFFS than patients at low risk (p = 0.000). The LRR pattern revealed significantly high incidences of recurrent disease at the supraclavicular and cervical sites, mediastinum (above the carina), and anastomosis. CONCLUSIONS: Our predictive model was able to distinguish between patients at high risk for LRR and patients at low risk for LRR. LRR primarily involved the upper thorax and this area must be considered in future study designs for radical trimodality treatment.
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Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/cirugía , Escisión del Ganglio Linfático/métodos , Recurrencia Local de Neoplasia , Adulto , Anciano , Área Bajo la Curva , Carcinoma de Células Escamosas/metabolismo , Supervivencia sin Enfermedad , Neoplasias Esofágicas/metabolismo , Esofagectomía , Femenino , Estudios de Seguimiento , Humanos , Receptores de Hialuranos/metabolismo , Linfocitos Infiltrantes de Tumor , Masculino , Mediastino , Persona de Mediana Edad , Cuello , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Curva ROC , Factores de Riesgo , Factores de TiempoRESUMEN
Some somatic illnesses such as peripheral tumours can present with psychiatric symptoms. Many of these are characterized by changes in biomarkers related to the inflammation or immune response. Here, we describe a multispectral imaging protocol that can be used to phenotype immune and other cell types through simultaneous imaging of multiple proteins in sections of peripheral solid tumours and other tissues. This approach can also be used to assess the spatial organization of these cells within the tissue.
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Fluorometría/métodos , Inmunofenotipificación/métodos , Trastornos Mentales/inmunología , Biomarcadores , Colorantes Fluorescentes , Formaldehído , Humanos , Procesamiento de Imagen Asistido por Computador , Trastornos Mentales/diagnóstico , Microscopía Fluorescente/métodos , Proteínas de Neoplasias/análisis , Neoplasias/diagnóstico , Neoplasias/inmunología , Proteínas del Tejido Nervioso/análisis , Adhesión en Parafina , Proteómica/métodos , Coloración y Etiquetado/métodos , Fijación del TejidoRESUMEN
We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.
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Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Proteína Fosfatasa 2/metabolismo , Transducción de Señal/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Clorhidrato de Fingolimod , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , MicroARNs/metabolismo , Modelos Biológicos , Fosforilación/efectos de los fármacos , Glicoles de Propileno/farmacología , Proteína Quinasa C-alfa/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacología , Familia-src Quinasas/metabolismoRESUMEN
Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may provide a rationale for designing therapeutics targeting LSC-enriched cell populations.
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Citometría de Flujo/métodos , Leucemia Mieloide Aguda/genética , Células Madre Neoplásicas/citología , Transducción de Señal/genética , Tirosina Quinasa 3 Similar a fms/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD/genética , Antígenos CD/inmunología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Citocinas/metabolismo , Humanos , Imidazoles/farmacología , Espectrometría de Masas/métodos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinolinas/farmacología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Coloración y Etiquetado , Factor de Células Madre/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Chromosomal region maintenance 1 (CRM1) is a nuclear export receptor recognizing proteins bearing a leucine-rich nuclear export signal. CRM1 is involved in nuclear export of tumor suppressors such as p53. We investigated the prognostic significance of CRM1 in acute myeloid leukemia (AML) and effects of a novel small-molecule selective inhibitor of CRM1. CRM1 protein expression was determined in 511 newly diagnosed AML patients and was correlated with mouse double minute 2 (MDM2) and p53 levels. High CRM1 expression was associated with short survival of patients and remained an adverse prognostic factor in multivariate analysis. CRM1 inhibitor KPT-185 induced mainly full-length p53 and apoptosis in a p53-dependent manner, whereas inhibition of proliferation was p53 independent. Patient samples with p53 mutations showed low sensitivity to KPT-185. Nuclear retention of p53 induced by CRM1 inhibition synergized with increased levels of p53 induced by MDM2 inhibition in apoptosis induction. KPT-185 and Nutlin-3a, alone and in combination, induced synergistic apoptosis in patient-derived CD34(+)/CD38(-) AML, but not in normal progenitor cells. Data suggest that CRM1 exerts an antiapoptotic function and is highly prognostic in AML. We propose a novel combinatorial approach for the therapy of AML, aimed at maximal activation of p53-mediated apoptosis by concomitant MDM2 and CRM1 inhibition.
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Acrilatos/uso terapéutico , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor , Carioferinas/antagonistas & inhibidores , Carioferinas/fisiología , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/fisiología , Triazoles/uso terapéutico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/fisiología , Células Cultivadas , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/genética , Femenino , Células HL-60 , Humanos , Carioferinas/genética , Leucemia Mieloide Aguda/genética , Masculino , Terapia Molecular Dirigida , Pronóstico , Receptores Citoplasmáticos y Nucleares/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Células U937 , Proteína Exportina 1RESUMEN
Although myeloid-derived immune cells can be dispersed throughout the tumor microenvironment (TME), anti-tumor effector cells are confined to the perivascular space. Here, we present a protocol to quantify immune cell distribution from tumor vasculature to its glioma microenvironment on sequential immunofluorescence multiplex images. We describe steps for sequential immunofluorescence multiplex staining, image generation, and storage. We then detail the procedures for tissue, vessel, and nuclei segmentation; cell phenotyping; data extraction; and training using RStudio and Spyder.
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Técnica del Anticuerpo Fluorescente , Glioma , Microambiente Tumoral , Microambiente Tumoral/inmunología , Glioma/inmunología , Glioma/patología , Glioma/irrigación sanguínea , Humanos , Técnica del Anticuerpo Fluorescente/métodos , Animales , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/irrigación sanguínea , Procesamiento de Imagen Asistido por Computador/métodos , RatonesRESUMEN
PURPOSE: Sinonasal undifferentiated carcinoma (SNUC) is a rare, aggressive malignancy of the sinonasal cavity with poor prognosis and limited treatment options. To investigate the potential for SNUC sensitivity to combinatory immunotherapy, we performed in vitro studies with SNUC cell lines and used multi-spectral immunofluorescence to characterize the in vivo patient SNUC tumor immune microenvironment (TIME). EXPERIMENTAL DESIGN: Human-derived SNUC cell lines were used for in vitro studies of tumor cell susceptibility to natural killer (NK) cell-based immunotherapeutic strategies. Tumor samples from 14 treatment naïve SNUC patients were examined via multi-spectral immunofluorescence and clinical correlations assessed. RESULTS: Anti-PD-L1 blockade enhanced NK cell lysis of SNUC cell lines â¼5.4 fold (P ≤ 0.0001). This effect was blocked by a CD16 neutralizing antibody demonstrating activity through an antibody-dependent cellular cytotoxicity (ADCC) mediated pathway. ADCC-dependent lysis of SNUC cells was further enhanced by upregulation of PD-L1 on tumor cells by exogenous interferon-gamma (IFN-γ) administration or interleukin-15 (IL-15) stimulated IFN-γ release from NK cells. Combination treatment with anti-PD-L1 blockade and IL-15 superagonism enhanced NK-cell killing of SNUC cells 9.6-fold (P ≤ 0.0001). Untreated SNUC patient tumor samples were found to have an NK cell infiltrate and PD-L1+ tumor cells at a median of 5.4 cells per mm2. A striking 55.7-fold increase in CKlow tumor cell/NK cell interactions was observed in patients without disease recurrence after treatment (P = 0.022). Patients with higher CD3+CD8+ in the stroma had a significantly improved 5-year overall survival (P = 0.0029) and a significant increase in CKlow tumor cell/CD8+ cytotoxic T cell interactions was noted in long-term survivors (P = 0.0225). CONCLUSION: These data provide the pre-clinical rationale for ongoing investigation into combinatory immunotherapy approaches for SNUC.
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Background & Aims: Clinical trials for reducing fibrosis in steatotic liver disease (SLD) have targeted macrophages with variable results. We evaluated intrahepatic macrophages in patients with SLD to determine if activity scores or fibrosis stages influenced phenotypes and expression of druggable targets, such as CCR2 and galectin-3. Methods: Liver biopsies from controls or patients with minimal or advanced fibrosis were subject to gene expression analysis using nCounter to determine differences in macrophage-related genes (n = 30). To investigate variability among individual patients, we compared additional biopsies by staining them with multiplex antibody panels (CD68/CD14/CD16/CD163/Mac387 or CD163/CCR2/galectin-3/Mac387) followed by spectral imaging and spatial analysis. Algorithms that utilize deep learning/artificial intelligence were applied to create cell cluster plots, phenotype profile maps, and to determine levels of protein expression (n = 34). Results: Several genes known to be pro-fibrotic (e.g. CD206, TREM2, CD163, and ARG1) showed either no significant differences or significantly decreased with advanced fibrosis. Although marked variability in gene expression was observed in individual patients with cirrhosis, several druggable targets and their ligands (e.g. CCR2, CCR5, CCL2, CCL5, and LGALS3) were significantly increased when compared to patients with minimal fibrosis. Antibody panels identified populations that were significantly increased (e.g. Mac387+), decreased (e.g. CD14+), or enriched (e.g. interactions of Mac387) in patients that had progression of disease or advanced fibrosis. Despite heterogeneity in patients with SLD, several macrophage phenotypes and druggable targets showed a positive correlation with increasing NAFLD activity scores and fibrosis stages. Conclusions: Patients with SLD have markedly varied macrophage- and druggable target-related gene and protein expression in their livers. Several patients had relatively high expression, while others were like controls. Overall, patients with more advanced disease had significantly higher expression of CCR2 and galectin-3 at both the gene and protein levels. Impact and implications: Appreciating individual differences within the hepatic microenvironment of patients with SLD may be paramount to developing effective treatments. These results may explain why such a small percentage of patients have responded to macrophage-targeting therapies and provide additional support for precision medicine-guided treatment of chronic liver diseases.
RESUMEN
Most platforms used for the molecular reconstruction of the tumor-immune microenvironment (TIME) of a solid tumor fail to explore the spatial context of the three-dimensional (3D) space of the tumor at a single-cell resolution, and thus lack information about cell-cell or cell-extracellular matrix (ECM) interactions. To address this issue, a pipeline which integrated multiplex spatially resolved multi-omics platforms was developed to identify crosstalk signaling networks among various cell types and the ECM in the 3D TIME of two FFPE (formalin-fixed paraffin embedded) gynecologic tumor samples. These platforms include non-targeted mass spectrometry imaging (glycans, metabolites, and peptides) and Stereo-seq (spatial transcriptomics) and targeted seqIF (IHC proteomics). The spatially resolved imaging data in a two- and three-dimensional space demonstrated various cellular neighborhoods in both samples. The collection of spatially resolved analytes in a voxel (3D pixel) across serial sections of the tissue was also demonstrated. Data collected from this analytical pipeline were used to construct spatial 3D maps with single-cell resolution, which revealed cell identity, activation, and energized status. These maps will provide not only insights into the molecular basis of spatial cell heterogeneity in the TIME, but also novel predictive biomarkers and therapeutic targets, which can improve patient survival rates.