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Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer, a disease with dismal overall survival. Advances in treatment are hindered by a lack of preclinical models. Here, we show how a personalized organotypic "avatar" created from resected tissue allows spatial and temporal reporting on a complete in situ tumor microenvironment and mirrors clinical responses. Our perfusion culture method extends tumor slice viability, maintaining stable tumor content, metabolism, stromal composition, and immune cell populations for 12 days. Using multiplexed immunofluorescence and spatial transcriptomics, we identify immune neighborhoods and potential for immunotherapy. We used avatars to assess the impact of a preclinically validated metabolic therapy and show recovery of stromal and immune phenotypes and tumor redifferentiation. To determine clinical relevance, we monitored avatar response to gemcitabine treatment and identify a patient avatar-predictable response from clinical follow-up. Thus, avatars provide valuable information for syngeneic testing of therapeutics and a truly personalized therapeutic assessment platform for patients.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Medicina de Precisión , Microambiente Tumoral , Animales , Humanos , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Desoxicitidina/farmacología , Gemcitabina , Inmunoterapia/métodos , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/terapia , Medicina de Precisión/métodos , Microambiente Tumoral/inmunologíaRESUMEN
We conducted this study aimed to examine the impact of evidence-based nursing interventions on postoperative wound pain and complications after surgery for finger tendon injury. A total of 86 patients treated for finger tendon injuries at our hospital from January 2021 to October 2023 were selected and randomly divided into an experimental group and a control group. The control group received conventional nursing care, while the experimental group received evidence-based nursing interventions. The study compared the postoperative wound pain intensity, incidence of complications and patient satisfaction with nursing care between the two groups. The analysis revealed that compared with conventional care, evidence-based nursing interventions significantly reduced the level of wound pain (p = 0.034) and the incidence of complications (4.65% vs. 18.60%, p = 0.043). It also increased patient satisfaction with the nursing care (97.67% vs. 83.72%, p = 0.026). The study indicates that the application of evidence-based nursing interventions for patients with finger tendon injuries can reduce postoperative wound pain, decrease the incidence of complications and enhance patient satisfaction with nursing care.
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Traumatismos de los Dedos , Traumatismos de los Tendones , Humanos , Enfermería Basada en la Evidencia , Traumatismos de los Dedos/cirugía , Dedos , Dolor Postoperatorio/terapia , Traumatismos de los Tendones/cirugíaRESUMEN
Evidence has revealed that DDOST plays an important role in cancer development and progression. However, there are no reports on functions of DDOST in cervical tumorigenesis. Hence, we investigated the relationship of DDOST with prognosis, mutation, promoter methylation, immune cell infiltration, and drug sensitivity using bioinformatics techniques. Our results demonstrated that DDOST was significantly upregulated in a variety of tumor types and correlated with poor prognosis, including cervical cancer. Cox regression analysis dissected that high DDOST expression was associated with poor survival in cervical cancer patients. Immune infiltration analysis defined that DDOST was negatively correlated with CD8 T cells and NK cells. Strikingly, the sensitivity to multiple drugs was negatively correlated with the expression of DDOST. Therefore, our findings uncovered that DDOST could play an essential role in the tumor microenvironment and tumor immune regulation in cervical cancer, which indicated that DDOST could be a useful biomarker for prognosis and a potential therapeutic target for cancer treatment.
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Objectives: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a 'preeclampsia-like syndrome'. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection. Methods: We utilised whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic (n = 9). Results: Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS-CoV-2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS-CoV-2 samples compared to uninfected controls. Conclusions: Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection.
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The utilization of single-cell resolved spatial transcriptomics to delineate immune responses during SARS-CoV-2 infection was able to identify M1 macrophages to have elevated expression of IFI27 in areas of infection.
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COVID-19 , Humanos , SARS-CoV-2 , Perfilación de la Expresión Génica , Macrófagos , Proteínas de la MembranaRESUMEN
Radiotherapy is still the recommended treatment for cervical cancer. However, radioresistance and radiation-induced side effects remain one of the biggest clinical problems. Selenium (Se) has been confirmed to exhibit radiation-enhancing effects for cancer treatment. However, Se species dominate the biological activities and which form of Se possesses better radiosensitizing properties and radiation safety remains elusive. Here, different Se species (the valence state of Se ranged from - 2, 0, +4 to + 6) synergy screen was carried out to identify the potential radiosensitizing effects and radiation safety of Se against cervical cancer. We found that the therapeutic effects varied with the changes in the Se valence state. Sodium selenite (+4) displayed strong cancer-killing effects but also possessed severe cytotoxicity. Sodium selenate (+6) neither enhanced the killing effects of X-ray nor possessed anticancer activity by its alone treatment. Although nano-selenium (0), especially Let-SeNPs, has better radiosensitizing activity, the - 2 organic Se, such as selenadiazole derivative SeD (-2) exhibited more potent anticancer effects and possessed a higher safe index. Overall, the selected Se drugs were able to synergize with X-ray to inhibit cell growth, clone formation, and cell migration by triggering G2/M phase arrest and apoptosis, and SeD (-2) was found to exhibit more potent enhancing capacity. Further mechanism studies showed that SeD mediated p53 pathway activation by inducing DNA damage through promoting ROS production. Additionally, SeD combined with X-ray therapy can induce an anti-tumor immune response in vivo. More importantly, SeD combined with X-ray significantly inhibited the liver metastasis of tumor cells and alleviated the side effects caused by radiation therapy in tumor-bearing mice. Taken together, this study demonstrates the radiosensitization and radiation safety effects of different Se species, which may shed light on the application of such Se-containing drugs serving as side effects-reducing agents for cervical cancer radiation treatment.
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Neoplasias Hepáticas , Fármacos Sensibilizantes a Radiaciones , Selenio , Neoplasias del Cuello Uterino , Humanos , Femenino , Ratones , Animales , Selenio/farmacología , Selenio/uso terapéutico , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/radioterapia , Proteína p53 Supresora de Tumor , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológicoRESUMEN
The SARS-CoV-2 (COVID-19) virus has caused a devastating global pandemic of respiratory illness. To understand viral pathogenesis, methods are available for studying dissociated cells in blood, nasal samples, bronchoalveolar lavage fluid and similar, but a robust platform for deep tissue characterization of molecular and cellular responses to virus infection in the lungs is still lacking. We developed an innovative spatial multi-omics platform to investigate COVID-19-infected lung tissues. Five tissue-profiling technologies were combined by a novel computational mapping methodology to comprehensively characterize and compare the transcriptome and targeted proteome of virus infected and uninfected tissues. By integrating spatial transcriptomics data (Visium, GeoMx and RNAScope) and proteomics data (CODEX and PhenoImager HT) at different cellular resolutions across lung tissues, we found strong evidence for macrophage infiltration and defined the broader microenvironment surrounding these cells. By comparing infected and uninfected samples, we found an increase in cytokine signalling and interferon responses at different sites in the lung and showed spatial heterogeneity in the expression level of these pathways. These data demonstrate that integrative spatial multi-omics platforms can be broadly applied to gain a deeper understanding of viral effects on cellular environments at the site of infection and to increase our understanding of the impact of SARS-CoV-2 on the lungs.
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In vitro, ACE2 translocates to the nucleus to induce SARS-CoV-2 replication. Here, using digital spatial profiling of lung tissues from SARS-CoV-2-infected golden Syrian hamsters, we show that a specific and selective peptide inhibitor of nuclear ACE2 (NACE2i) inhibits viral replication two days after SARS-CoV-2 infection. Moreover, the peptide also prevents inflammation and macrophage infiltration, and increases NK cell infiltration in bronchioles. NACE2i treatment increases the levels of the active histone mark, H3K27ac, restores host translation in infected hamster bronchiolar cells, and leads to an enrichment in methylated ACE2 in hamster bronchioles and lung macrophages, a signature associated with virus protection. In addition, ACE2 methylation is increased in myeloid cells from vaccinated patients and associated with reduced SARS-CoV-2 spike protein expression in monocytes from individuals who have recovered from infection. This protective epigenetic scarring of ACE2 is associated with a reduced latent viral reservoir in monocytes/macrophages and enhanced immune protection against SARS-CoV-2. Nuclear ACE2 may represent a therapeutic target independent of the variant and strain of viruses that use the ACE2 receptor for host cell entry.
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COVID-19 , SARS-CoV-2 , Cricetinae , Animales , Humanos , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/metabolismo , Pulmón/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Péptidos/metabolismo , Epigénesis GenéticaRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to present with pulmonary and extra-pulmonary organ complications. In comparison with the 2009 pandemic (pH1N1), SARS-CoV-2 infection is likely to lead to more severe disease, with multi-organ effects, including cardiovascular disease. SARS-CoV-2 has been associated with acute and long-term cardiovascular disease, but the molecular changes that govern this remain unknown. In this study, we investigated the host transcriptome landscape of cardiac tissues collected at rapid autopsy from seven SARS-CoV-2, two pH1N1, and six control patients using targeted spatial transcriptomics approaches. Although SARS-CoV-2 was not detected in cardiac tissue, host transcriptomics showed upregulation of genes associated with DNA damage and repair, heat shock, and M1-like macrophage infiltration in the cardiac tissues of COVID-19 patients. The DNA damage present in the SARS-CoV-2 patient samples, were further confirmed by γ-H2Ax immunohistochemistry. In comparison, pH1N1 showed upregulation of interferon-stimulated genes, in particular interferon and complement pathways, when compared with COVID-19 patients. These data demonstrate the emergence of distinct transcriptomic profiles in cardiac tissues of SARS-CoV-2 and pH1N1 influenza infection supporting the need for a greater understanding of the effects on extra-pulmonary organs, including the cardiovascular system of COVID-19 patients, to delineate the immunopathobiology of SARS-CoV-2 infection, and long term impact on health.
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COVID-19 , Enfermedades Cardiovasculares , Humanos , SARS-CoV-2 , Transcriptoma , InterferonesRESUMEN
The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.
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Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability in the USA. The progression of neuronal damage after stroke is recognized to be a complex integration of glia, neurons, and the surrounding extracellular matrix, therefore potential treatments must target the detrimental effects created by these interactions. In this study, we examined the spatial cellular and neuroinflammatory mechanisms occurring early after ischemic stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. Male C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and sacrificed at 3 days post-ischemia. Spatial distinction of the ipsilateral hemisphere was studied according to the regions of interest: the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in comparison to the contralateral hemisphere. We demonstrated that the ipsilateral hemisphere initiates distinct spatial regulatory proteomic profiles with DSP technology that can be identified consistently with the immunohistochemical markers, FJB, GFAP, and Iba-1. The core border profile demonstrated an induction of neuronal death, apoptosis, autophagy, immunoreactivity, and early degenerative proteins. Most notably, the core border resulted in a decrease of the neuronal proteins Map2 and NeuN; an increase in the autophagy proteins BAG3 and CTSD; an increase in the microglial and peripheral immune invasion proteins Iba1, CD45, CD11b, and CD39; and an increase in the neurodegenerative proteins BACE1, APP, amyloid ß 1-42, ApoE, and hyperphosphorylated tau protein S-199. The peri-infarct region demonstrated increased astrocytic, immunoreactivity, apoptotic, and neurodegenerative proteomic profiles, with an increase in BAG3, GFAP, and hyperphosphorylated tau protein S-199. The PiNT region displayed minimal changes compared to the contralateral cortex with only an increase in GFAP. In this study, we showed that mechanisms known to be associated with stroke, such as apoptosis and inflammation, occur in distinct spatial domains of the injured brain following ischemia. We also demonstrated the dysregulation of specific autophagic pathways that may lead to neurodegeneration in peri-infarct brain tissues. Taken together, these data suggest that identifying post-ischemic mechanisms occurring in a spatiotemporal manner may lead to more precise targets for successful therapeutic interventions to treat stroke.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Ratones , Masculino , Proteínas tau/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteómica , Ácido Aspártico Endopeptidasas/metabolismo , Neuronas/metabolismo , Accidente Cerebrovascular/metabolismo , Isquemia Encefálica/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/metabolismo , Ratones Endogámicos C57BL , Análisis Espacial , Modelos Animales de EnfermedadRESUMEN
Cervical cancer (CC) ranks as the second most frequent tumor in women, and CC stem cells have been vital in the tumorigenesis of CC. Recently, the metastasis- associated in colon cancer 1 (MACC1) gene was proven to be a promising biomarker of CC. However, the role and mechanism of MACC1 in CC remain undetermined. Expressions of MACC1 were estimated by qRT-PCR, immunohistochemistry, and Western blot assays in cervical cancer tissues and cells. Three siRNAs were generated to knockdown expressions of MACC1 in CC cells. After knockdown of MACC1 or/and Colivelin treatment, cell migration, invasion, apoptosis, and stemness were evaluated through a series of functional experiments including Transwell, flow cytometry, Hoechst staining, and sphere-formation assays. MACC1 was found to express more highly in CC tissues in comparison with corresponding non-tumor tissues at both mRNA and protein levels. Functionally, the knocking- down of MACC1 significantly repressed migration and invasion, and induced apoptosis of CC cells. Also, knockdown of MACC1 was discovered to suppress sphere-formation of CC cells and downregulate OCT4 and Nanog. It was proved that knockdown of MACC1 had a significant blocking effect on the AKT/STAT3 pathway. In addition, we verified that treatment with the STAT3 activator (Colivelin) had significant reversal effects on the malignant behaviors of CC cells and CC stemness. Our study concluded that MACC1 might be a novel regulator of CC by regulating the AKT/STAT3 pathway to change the migration, invasion, apoptosis, and cancer stemness of CC cells.
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Células Madre Neoplásicas/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Apoptosis , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Invasividad Neoplásica , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Neoplasias del Cuello Uterino/genéticaRESUMEN
BACKGROUND: Endometriosis (EMs) is a benign, but potential metastatic, gynecological disease. Our current study aims to examine whether all-trans retinoic acid (ATRA) inhibits the epithelial-to-mesenchymal transition (EMT) of endometriotic stromal stem cells, as well as to explore the mechanisms involved, especially the role of IL-6 played in. METHODS: Cell clonogenic capacity was examined by the low-density clonogenicity assay. Cell differentiation capacity was assessed by in vitro differentiation. The level of IL-6 was measured by the ELISA assay. Migration and invasion abilities were measured using the transwell assay. Western blot and RT-qPCR were performed to detect EMT-related genes and proteins. RESULTS: Large endometriotic stromal colony forming units (CFUs) could be regarded as the enrichment sets of endometriotic stromal stem cells. They maintained a higher potential for self-renewal, proliferation, invasion, and EMT, along with up-regulated IL-6. After ATRA treatment, the expression of IL-6 was significantly reduced, accompanied by a decrease in the migration, invasion, and EMT of large endometriotic stromal CFUs. In addition, the inhibition of ATRA was mediated by IL-6. CONCLUSIONS: Our study showed that one of the therapeutic effects of ATRA on EMs through its modulation in EMT of large endometriotic stromal CFUs. ATRA may be a promising therapeutic strategy aimed at IL-6 for the stem-cell treatment of EMs.
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Endometriosis , Regulación hacia Abajo , Endometriosis/tratamiento farmacológico , Transición Epitelial-Mesenquimal , Femenino , Humanos , Interleucina-6/genética , Tretinoina/farmacologíaRESUMEN
Anosmia, the loss of smell, is a common and often the sole symptom of COVID-19. The onset of the sequence of pathobiological events leading to olfactory dysfunction remains obscure. Here, we have developed a postmortem bedside surgical procedure to harvest endoscopically samples of respiratory and olfactory mucosae and whole olfactory bulbs. Our cohort of 85 cases included COVID-19 patients who died a few days after infection with SARS-CoV-2, enabling us to catch the virus while it was still replicating. We found that sustentacular cells are the major target cell type in the olfactory mucosa. We failed to find evidence for infection of olfactory sensory neurons, and the parenchyma of the olfactory bulb is spared as well. Thus, SARS-CoV-2 does not appear to be a neurotropic virus. We postulate that transient insufficient support from sustentacular cells triggers transient olfactory dysfunction in COVID-19. Olfactory sensory neurons would become affected without getting infected.
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Autopsia/métodos , COVID-19/mortalidad , COVID-19/virología , Bulbo Olfatorio/virología , Mucosa Olfatoria/virología , Mucosa Respiratoria/virología , Anciano , Anosmia , COVID-19/fisiopatología , Endoscopía/métodos , Femenino , Glucuronosiltransferasa/biosíntesis , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Trastornos del Olfato , Neuronas Receptoras Olfatorias/metabolismo , Sistema Respiratorio , SARS-CoV-2 , OlfatoRESUMEN
Discovering compounds with anti-cervical cancer effect and clarifying their targets will help promoting the precise treatment of cervical cancer. The present study intended to clarify the effect of osthole on cervical cancer cells, and to explore the possibility of DCLK1 as its target. Annexin V-PE staining and flow cytometry methods were used to determine cell apoptosis. Meanwhile, apoptosis related biomarkers were probed by immunoblotting. The MTT assay was employed to study the effect of osthole in combined with or without LRRK2-IN-1 (a DCLK1 inhibitor) on cell proliferation. Then, combination index was determined. To examine the interaction of osthole with DCLK1, molecular docking was carried out. Based on the biological database from cBioPortal, the association between DCLK1 and clinical manifestations of cervical cancer were evaluated. The results showed that osthole can significantly induce apoptosis of HeLa and Me-180. When combined with LRRK2-IN-1, the effect of osthole on cell proliferation was antagonized, suggesting that it might competitive binding to DCLK1. Furthermore, molecular docking showed that osthole interacts with Val468 residues of DCLK1 to form hydrogen bonds. The analysis of database showed that DCLK1 frequently mutant and deleted in cervical cancer, and is related to cell survival, tumor progression and recurrence. However, no obvious correlations were found between DCLK1 and lymphatic metastasis/differentiation. In conclusion, osthole significantly inhibits the survival of cervical cancer cells. It's probably target DCLK1 mechanistically via interacting with Val468. DCLK1 could be a potential therapeutic target for cervical cancer.
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Neoplasias del Cuello Uterino , Línea Celular Tumoral , Proliferación Celular , Cumarinas/farmacología , Quinasas Similares a Doblecortina , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Simulación del Acoplamiento Molecular , Proteínas Serina-Treonina Quinasas/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genéticaRESUMEN
We aimed to investigate the biological roles of Argonaute 3 (AGO3) in cervical cancer. RNA profiles containing 306 cervical cancer tissues and 13 normal samples revealed that AGO3 was significantly up-regulated in cervical cancer, and the expression of AGO3 was negatively associated with the outcome of cervical cancer patients. Cell proliferation and transwell assays showed that the depletion of AGO3 markedly inhibited cervical cancer cell growth and mobility. Importantly, we detected that knockdown of AGO3 exerted suppressive effect on cellular behaviors via inactivating Wnt/ß-catenin signaling pathway. Collectively, we conclude that AGO3 is a novel tumor promoter in cervical cancer and has a potential to be a drug target and prognostic predictor of cervical cancer patients.
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Proteínas Argonautas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Argonautas/genética , Línea Celular , Ensayos de Migración Celular , Supervivencia Celular , Cuello del Útero/citología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Transducción de Señal , Proteínas Wnt/genética , beta Catenina/genéticaRESUMEN
BACKGROUND: In melanoma patients, microscopic tumor in the sentinel lymph-node biopsy (SLN) increases the risk of distant metastases, but the transition from tumor in the SLN to metastatic disease remains poorly understood. METHODS: Fluorescent staining for CD3, CD20, CD11c, and DNA was performed on SLN tissue and matching primary tumors. Regions of interest (ROI) were then chosen geometrically (e.g., tumor) or by fluorescent cell subset markers (e.g., CD11c). Each ROI was further analyzed using NanoString Digital Spatial Profiling high-resolution multiplex profiling. Digital counts for 59-panel immune-related proteins were collected and normalized to account for system variation and ROI area. RESULTS: Tumor regions of SLNs had variable infiltration of CD3 cells among patients. The patient with overall survival (OS) > 8 years had the most CD11c- and CD3-expressing cells infiltrating the SLN tumor region. All patients had CD11c (dendritic cell, DC) infiltration into the SLN tumor region. Selecting ROI by specific cell subtype, we compared protein expression of CD11c cells between tumor and non-tumor/normal tissue SLN regions. Known markers of DC activation such as CD86, HLA-DR, and OX40L were lowest on CD11c cells within SLN tumor for the patient with OS < 1 year and highest on the patient with OS > 8 years. CONCLUSION: We demonstrate the feasibility of profiling the protein expression of CD11c cells within the SLN tumor. Identifying early regulators of melanoma control when the disease is microscopically detected in the SLN is beneficial and requires follow-up studies in a larger cohort of patients.
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Metástasis Linfática/inmunología , Melanoma/inmunología , Biopsia del Ganglio Linfático Centinela/métodos , Microambiente Tumoral/inmunología , Femenino , Humanos , MasculinoRESUMEN
In this study, we investigated the effect of silybin on the pharmacokinetics of brexpiprazole and the underlying mechanism in rats. Two groups of animals received silybin at different doses (50 mg/kg, 25 mg/kg) for 2 weeks, while another group was given vehicle alone. After that, rats were intragastrically administrated with 2 mg/kg brexpiprazole. Then, the tail blood and liver tissues were collected from each rat at different time points. Brexpiprazole in serum was determined by an established UPLC-MS/MS assay. Finally, pharmacokinetic parameters of animals in each group were figured out. The results show that silybin remarkably changed the pharmacokinetic properties of brexpiprazole, especially at the highest dose. AUC and Cmax in the combination group with 50 mg/kg silybin were enhanced approximately 4 times as much as after a single dose of brexpiprazole, p < 0.05. Meanwhile, total liver protein of each sample was extracted, and was subjected to immunoblotting assay for probing CYP3A4 and CYP2D6. Therein CYP3A4 was significantly downregulated compared to the control group. Overall, silybin can increase blood concentration of brexpiprazole in rat by downregulating its main metabolic enzyme CYP3A4. Therefore, the maintenance dose of brexpiprazole should be decreased while co-treating with silybin.
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Antipsicóticos/farmacología , Sustancias Protectoras/farmacología , Quinolonas/farmacocinética , Silibina/farmacología , Tiofenos/farmacocinética , Animales , Antipsicóticos/administración & dosificación , Área Bajo la Curva , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Interacciones Farmacológicas , Masculino , Sustancias Protectoras/administración & dosificación , Ratas , Ratas Sprague-Dawley , Silibina/administración & dosificación , Espectrometría de Masas en TándemRESUMEN
WZ35 is a monocarbonyl analog of curcumin, which had been proved advantage over curcumin in chemical stability and antitumor activity. However, its pharmacokinetic profile has not been determined. In the present study, an ultraperformance liquid chromatography-tandem mass spectrometry assay was developed to detect concentration of WZ35 in rat plasma. Subsequently, pharmacokinetic study showed that the oral bioavailability of WZ35 is 10.56%. Cytochrome P450 (CYP450) plays a major role in metabolizing exogenous substance. The concentration of WZ35 was sharply decreased while incubating with microsome. It's indicated that WZ35 is a substrate of CYP450s. Molecular docking assay showed that WZ35 can combine with CYP2B6 and CYP2C9 to form much more stable complex. The lowest docking energy was generated in complex with CYP2E1. The inhibition of CYP450s by WZ35 was also evaluated. Pan inhibitions of WZ35 on rat CYP3A2, CYP2B1, CYP2C11, CYP2D1, and -CYP2E1 were observed by detecting probe substrates (midazolam, bupropion, tolbutamide, dextromethorphan, chlorzoxazone) and metabolites accordingly. On an average, 80% activities of enzymes were blocked. Mechanistically, the inhibitions of WZ35 on CYP3A2, CYP2B1, CYP2E1 were in a time-dependent manner according to the results of IC50 shift assay. The collective data demonstrated that the oral bioavailability of monocarbonyl analog of curcumin has significantly improved compared to curcumin. It's both the substrate and inhibitor of CYP450s through in a time-dependent mechanism.
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Antineoplásicos/farmacocinética , Curcumina/análogos & derivados , Curcumina/farmacocinética , Inhibidores Enzimáticos del Citocromo P-450/farmacocinética , Animales , Antineoplásicos/sangre , Disponibilidad Biológica , Inhibidores Enzimáticos del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/metabolismo , Masculino , Simulación del Acoplamiento Molecular , Ratas Sprague-DawleyRESUMEN
Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth. Current genetic ITH analysis methods do not preserve spatial context and may not detect rare subclones. Here, we address these shortfalls by developing and validating BaseScope-a novel mutation-specific RNA in situ hybridization assay. We target common point mutations in the BRAF, KRAS and PIK3CA oncogenes in archival colorectal cancer samples to precisely map the spatial and morphological context of mutant subclones. Computational modeling suggests that subclones must arise sufficiently early, or carry a considerable fitness advantage, to form large or spatially disparate subclones. Examples of putative treatment-resistant cells isolated in small topographical areas are observed. The BaseScope assay represents a significant technical advance for in situ mutation detection that provides new insight into tumor evolution, and could have ramifications for selecting patients for treatment.