RESUMEN
Regenerative stem cell-like memory (TSCM) CD8+ T cells persist longer and produce stronger effector functions. We found that MEK1/2 inhibition (MEKi) induces TSCM that have naive phenotype with self-renewability, enhanced multipotency and proliferative capacity. This is achieved by delaying cell division and enhancing mitochondrial biogenesis and fatty acid oxidation, without affecting T cell receptor-mediated activation. DNA methylation profiling revealed that MEKi-induced TSCM cells exhibited plasticity and loci-specific profiles similar to bona fide TSCM isolated from healthy donors, with intermediate characteristics compared to naive and central memory T cells. Ex vivo, antigenic rechallenge of MEKi-treated CD8+ T cells showed stronger recall responses. This strategy generated T cells with higher efficacy for adoptive cell therapy. Moreover, MEKi treatment of tumor-bearing mice also showed strong immune-mediated antitumor effects. In conclusion, we show that MEKi leads to CD8+ T cell reprogramming into TSCM that acts as a reservoir for effector T cells with potent therapeutic characteristics.
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Antineoplásicos/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Inmunoterapia Adoptiva , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias/terapia , Células Madre/citología , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Ciclo Celular/efectos de los fármacos , Humanos , Memoria Inmunológica/inmunología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/fisiología , Microambiente TumoralRESUMEN
Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.
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Dinoprostona/análogos & derivados , Dinoprostona/metabolismo , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Grasa Intraabdominal/inmunología , Linfocitos T Reguladores/enzimología , Linfocitos T Reguladores/inmunología , Células 3T3 , Animales , Línea Celular , Diabetes Mellitus Tipo 2/metabolismo , Células HEK293 , Homeostasis/inmunología , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Resistencia a la Insulina/genética , Grasa Intraabdominal/citología , Células Jurkat , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Noqueados , Factor de Transcripción STAT5/metabolismoRESUMEN
While radical prostatectomy remains the mainstay of prostate cancer (PCa) treatment, 20 to 40% of patients develop postsurgical biochemical recurrence (BCR). A particularly challenging clinical cohort includes patients with intermediate-risk disease whose risk stratification would benefit from advanced approaches that complement standard-of-care diagnostic tools. Here, we show that imaging tumor lactate using hyperpolarized 13C MRI and spatial metabolomics identifies BCR-positive patients in two prospective intermediate-risk surgical cohorts. Supported by spatially resolved tissue analysis of established glycolytic biomarkers, this study provides the rationale for multicenter trials of tumor metabolic imaging as an auxiliary tool to support PCa treatment decision-making.
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Antígeno Prostático Específico , Neoplasias de la Próstata , Masculino , Humanos , Antígeno Prostático Específico/análisis , Ácido Láctico , Estudios Prospectivos , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/cirugía , Próstata/patología , Prostatectomía/métodos , Recurrencia Local de Neoplasia/diagnóstico por imagen , Recurrencia Local de Neoplasia/patología , Estudios RetrospectivosRESUMEN
Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, rapid acute induction of PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for this specialized PGR function in ovulation is poorly understood. We have assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrate that stimulating ovulation rapidly reprograms chromatin accessibility in two-thirds of sites, correlating with altered gene expression. An ovary-specific PGR action involving interaction with RUNX transcription factors was observed with 70% of PGR-bound regions also bound by RUNX1. These transcriptional complexes direct PGR binding to proximal promoter regions. Additionally, direct PGR binding to the canonical NR3C motif enable chromatin accessibility. Together these PGR actions mediate induction of essential ovulatory genes. Our findings highlight a novel PGR transcriptional mechanism specific to ovulation, providing new targets for infertility treatments or new contraceptives that block ovulation.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal , Regulación de la Expresión Génica , Receptores de Progesterona , Transcripción Genética , Animales , Femenino , Ratones , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Mamíferos/genética , Ratones Noqueados , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismoRESUMEN
BACKGROUND/OBJECTIVE: To explore the anti-tumour activity of combining AKT inhibition and docetaxel in PTEN protein null and WT prostate tumours. METHODS: Mechanisms associated with docetaxel capivasertib treatment activity in prostate cancer were examined using a panel of in vivo tumour models and cell lines. RESULTS: Combining docetaxel and capivasertib had increased activity in PTEN null and WT prostate tumour models in vivo. In vitro short-term docetaxel treatment caused cell cycle arrest in the majority of cells. However, a sub-population of docetaxel-persister cells did not undergo G2/M arrest but upregulated phosphorylation of PI3K/AKT pathway effectors GSK3ß, p70S6K, 4E-BP1, but to a lesser extent AKT. In vivo acute docetaxel treatment induced p70S6K and 4E-BP1 phosphorylation. Treating PTEN null and WT docetaxel-persister cells with capivasertib reduced PI3K/AKT pathway activation and cell cycle progression. In vitro and in vivo it reduced proliferation and increased apoptosis or DNA damage though effects were more marked in PTEN null cells. Docetaxel-persister cells were partly reliant on GSK3ß as a GSK3ß inhibitor AZD2858 reversed capivasertib-induced apoptosis and DNA damage. CONCLUSION: Capivasertib can enhance anti-tumour effects of docetaxel by targeting residual docetaxel-persister cells, independent of PTEN status, to induce apoptosis and DNA damage in part through GSK3ß.
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Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-akt , Pirimidinas , Pirroles , Masculino , Humanos , Docetaxel/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/farmacología , Transducción de Señal , Apoptosis , Fosfatidilinositol 3-Quinasas/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Línea Celular Tumoral , Puntos de Control de la Fase G2 del Ciclo Celular , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Fosfohidrolasa PTEN/metabolismoRESUMEN
Patients with prostate cancer frequently show resistance to androgen-deprivation therapy, a condition known as castration-resistant prostate cancer (CRPC). Acquiring a better understanding of the mechanisms that control the development of CRPC remains an unmet clinical need. The well-established dependency of cancer cells on the tumour microenvironment indicates that the microenvironment might control the emergence of CRPC. Here we identify IL-23 produced by myeloid-derived suppressor cells (MDSCs) as a driver of CRPC in mice and patients with CRPC. Mechanistically, IL-23 secreted by MDSCs can activate the androgen receptor pathway in prostate tumour cells, promoting cell survival and proliferation in androgen-deprived conditions. Intra-tumour MDSC infiltration and IL-23 concentration are increased in blood and tumour samples from patients with CRPC. Antibody-mediated inactivation of IL-23 restored sensitivity to androgen-deprivation therapy in mice. Taken together, these results reveal that MDSCs promote CRPC by acting in a non-cell autonomous manner. Treatments that block IL-23 can oppose MDSC-mediated resistance to castration in prostate cancer and synergize with standard therapies.
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Interleucina-23/antagonistas & inhibidores , Interleucina-23/metabolismo , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/terapia , Antagonistas de Receptores Androgénicos/farmacología , Antagonistas de Receptores Androgénicos/uso terapéutico , Andrógenos/deficiencia , Animales , Benzamidas , Proliferación Celular , Supervivencia Celular , Humanos , Interleucina-23/sangre , Interleucina-23/inmunología , Masculino , Ratones , Células Supresoras de Origen Mieloide/citología , Células Supresoras de Origen Mieloide/inmunología , Nitrilos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Feniltiohidantoína/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/sangre , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Receptores de Interleucina/metabolismo , Transducción de SeñalRESUMEN
AIMS/HYPOTHESIS: NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation. METHODS: We generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (ßp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (ßNEMOKO mice), as well as ßA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation-sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme. RESULTS: Rela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as ßp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in ßp65KO islets. CONCLUSIONS/INTERPRETATION: These data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.
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Intolerancia a la Glucosa , Factor de Transcripción ReIA , Animales , Humanos , Ratones , Cromatina , Glucosa , FN-kappa B/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
A significant number of babies present transiently with low protein kinase C zeta (PKCζ) levels in cord blood T cells (CBTC), associated with reduced ability to transition from a neonatal Th2 to a mature Th1 cytokine bias, leading to a higher risk of developing allergic sensitisation, compared to neonates whose T cells have 'normal' PKCζ levels. However, the importance of PKCζ signalling in regulating their differentiation from a Th2 to a Th1 cytokine phenotype propensity remains undefined. To define the role of PKCζ signalling in the regulation of CBTC differentiation from a Th2 to a Th1cytokine phenotype we have developed a neonatal T cell maturation model which enables the cells to develop to CD45RA- /CD45RO+ T cells while maintaining the Th2 immature cytokine bias, despite having normal levels of PKCζ. The immature cells were treated with phytohaemagglutinin, but in addition with phorbol 12-myristate 13-acetate (PMA), an agonist which does not activate PKCζ. This was compared to development in CBTC in which the cells were transfected to express constitutively active PKCζ. The lack of PKCζ activation by PMA was monitored by western blot for phospho-PKCζ and translocation from cell cytosol to the membrane by confocal microscopy. The findings demonstrate that PMA fails to activate PKCζ in CBTC. The data show that CBTC matured under the influence of the PKC stimulator, PMA, maintain a Th2 cytokine bias, characterised by robust IL-4 and minimal interferon gamma production (IFN-γ), and lack of expression of transcriptional factor, T-bet. This was also reflected in the production of a range of other Th2/Th1 cytokines. Interestingly, introduction of a constitutively active PKCζ mutant into CBTC promoted development towards a Th1 profile with high IFN-γ production. The findings demonstrate that PKCζ signalling is essential for the immature neonatal T cells to transition from a Th2 to a Th1 cytokine production bias.
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Interferón gamma , Células TH1 , Recién Nacido , Humanos , Interferón gamma/metabolismo , Células TH1/metabolismo , Sangre Fetal , Citocinas/metabolismo , Diferenciación Celular , Antígenos Comunes de Leucocito , Células Th2/metabolismoRESUMEN
The adenomatous polyposis coli (Apc) protein regulates diverse effector pathways essential for tissue homeostasis. Truncating oncogenic mutations in Apc removing its Wnt pathway and microtubule regulatory domains drives intestinal epithelia tumorigenesis. Exuberant cell proliferation is one well-established consequence of oncogenic Wnt pathway activity; however, the contribution of other deregulated molecular circuits to tumorigenesis has not been fully examined. Using in vivo and organoid models of intestinal epithelial tumorigenesis we found that Wnt pathway activity controls intestinal epithelial villi and crypt structure, morphological features lost upon Apc inactivation. Although the Wnt pathway target gene c-Myc (also known as Myc) has critical roles in regulating cell proliferation and tumorigenesis, Apc specification of intestinal epithelial morphology is independent of the Wnt-responsive Myc-335 (also known as Rr21) regulatory element. We further demonstrate that Apc inactivation disrupts the microtubule cytoskeleton and consequently localisation of organelles without affecting the distribution of the actin cytoskeleton and associated components. Our data indicates the direct control over microtubule dynamics by Apc through an independent molecular circuit. Our study stratifies three independent Apc effector pathways in the intestinal epithelial controlling: (1) proliferation, (2) microtubule dynamics and (3) epithelial morphology.This article has an associated First Person interview with the first author of the paper.
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Proteína de la Poliposis Adenomatosa del Colon , Vía de Señalización Wnt , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Carcinogénesis , Proliferación Celular/genética , Humanos , Mucosa Intestinal/metabolismo , Mutación/genética , Vía de Señalización Wnt/genéticaRESUMEN
High rates of drug-resistant tuberculosis in Ukraine suggest screening is necessary to mitigate public health hazards for host populations. A pathway was implemented in Wales and data prospectively collected Between 8 April and 21 December 2022. Of 5425 Ukrainian arrivals, notifications were received by TB teams on 2395 (44%) of whom 1955 (82%) were screened. The refugees were young (median age 30, IQR 14-41), and predominantly female (66.1%). Interferon- gamma release assay (IGRA) tests were positive in 112 (6.5%). One Case of active tuberculosis was identified (0.05%). Our data supports European guidelines that routine screening of this population is not recommended, but we remain uncertain as to the risks of this population going forwards.
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Tuberculosis Latente , Refugiados , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Femenino , Adulto , Masculino , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/epidemiología , Prueba de Tuberculina , Gales/epidemiología , Ensayos de Liberación de Interferón gamma , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tamizaje MasivoRESUMEN
Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.
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Ensamble y Desensamble de Cromatina/inmunología , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica , Proteínas de Unión a la Región de Fijación a la Matriz/inmunología , Autotolerancia , Linfocitos T Reguladores/inmunología , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Lentivirus , Activación de Linfocitos/efectos de los fármacos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/inmunología , MicroARNs/metabolismo , MicroARNs/farmacología , Interferencia de ARN , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Autotolerancia/efectos de los fármacos , Autotolerancia/genética , Autotolerancia/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Transducción GenéticaRESUMEN
OBJECTIVE: Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DESIGN: Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. RESULTS: Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. CONCLUSION: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.
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BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.
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Adenocarcinoma/patología , Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Neoplasias Pancreáticas/patología , Adenocarcinoma/genética , Adenocarcinoma/terapia , Biomarcadores , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Humanos , Terapia Molecular Dirigida , Organoides , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/tratamiento farmacológico , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Ratones , Imagen Multimodal , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral , GemcitabinaRESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as "long COVID", post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. METHODS: We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. RESULTS: Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. CONCLUSIONS: Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.
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COVID-19 , Anticuerpos Antivirales , COVID-19/complicaciones , Humanos , Sistema Inmunológico , SARS-CoV-2 , Síndrome Post Agudo de COVID-19RESUMEN
Toxicologic evaluation of new drug candidates routinely utilizes healthy animals. In oncology, there remains a limited understanding of the effects of novel test candidates in a diseased host. For vascular modulating agents (VMAs), an increased understanding of preclinical tumour-host interaction, and its potential to exacerbate or alleviate 'off-target' effects of anti-angiogenic administration, could aid in the prediction of adverse clinical outcomes in a defined cancer patient. We have previously reported that the implantation and growth of a range of human- and mouse-derived tumours leads to structural vascular and, potentially, functional signalling changes within host mouse endocrine tissues, indicating possible roles for tumour- and host-derived cytokines/growth factors and the liberation of myeloid-derived suppressor cells in this phenomenon. Here, we further demonstrate that the growth of the Calu-6 xenograft is associated with a resistance to VMA-induced mouse peripheral endocrine vascular rarefaction (toxicity), with potential functional impact, notably with respect to mixed tyrosine kinase inhibition. The pathogenesis of these findings indicates a potential role for both tumour- and host-derived basic fibroblast growth factor (bFGF), with associated upregulation in the intra-tumoural autotaxin-lysophosphatic acid signalling axis.
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Neoplasias , Neovascularización Patológica , Animales , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológicoRESUMEN
The burden of nosocomial SARS-CoV-2 infection remains poorly defined. We report on the outcomes of 2508 adults with molecularly-confirmed SARS-CoV-2 admitted across 18 major hospitals, representing over 60% of those hospitalised across Wales between 1 March and 1 July 2020. Inpatient mortality for nosocomial infection ranged from 38% to 42%, consistently higher than participants with community-acquired infection (31%-35%) across a range of case definitions. Those with hospital-acquired infection were older and frailer than those infected within the community. Nosocomial diagnosis occurred a median of 30 days following admission (IQR 21-63), suggesting a window for prophylactic or postexposure interventions, alongside enhanced infection control measures.
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COVID-19 , Infección Hospitalaria , Adulto , Infección Hospitalaria/epidemiología , Hospitales , Humanos , Estudios Retrospectivos , SARS-CoV-2 , Gales/epidemiologíaRESUMEN
An ever-increasing array of imaging technologies are being used in the study of complex biological samples, each of which provides complementary, occasionally overlapping information at different length scales and spatial resolutions. It is important to understand the information provided by one technique in the context of the other to achieve a more holistic overview of such complex samples. One way to achieve this is to use annotations from one modality to investigate additional modalities. For microscopy-based techniques, these annotations could be manually generated using digital pathology software or automatically generated by machine learning (including deep learning) methods. Here, we present a generic method for using annotations from one microscopy modality to extract information from complementary modalities. We also present a fast, general, multimodal registration workflow [evaluated on multiple mass spectrometry imaging (MSI) modalities, matrix-assisted laser desorption/ionization, desorption electrospray ionization, and rapid evaporative ionization mass spectrometry] for automatic alignment of complex data sets, demonstrating an order of magnitude speed-up compared to previously published work. To demonstrate the power of the annotation transfer and multimodal registration workflows, we combine MSI, histological staining (such as hematoxylin and eosin), and deep learning (automatic annotation of histology images) to investigate a pancreatic cancer mouse model. Neoplastic pancreatic tissue regions, which were histologically indistinguishable from one another, were observed to be metabolically different. We demonstrate the use of the proposed methods to better understand tumor heterogeneity and the tumor microenvironment by transferring machine learning results freely between the two modalities.
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Aprendizaje Profundo , Animales , Técnicas Histológicas , Ratones , Imagen Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Flujo de TrabajoRESUMEN
Clinical tissue specimens are often unscreened, and preparation of tissue sections for analysis by mass spectrometry imaging (MSI) can cause aerosolization of particles potentially carrying an infectious load. We here present a decontamination approach based on ultraviolet-C (UV-C) light to inactivate clinically relevant pathogens such as herpesviridae, papovaviridae human immunodeficiency virus, or SARS-CoV-2, which may be present in human tissue samples while preserving the biodistributions of analytes within the tissue. High doses of UV-C required for high-level disinfection were found to cause oxidation and photodegradation of endogenous species. Lower UV-C doses maintaining inactivation of clinically relevant pathogens to a level of increased operator safety were found to be less destructive to the tissue metabolome and xenobiotics. These doses caused less alterations of the tissue metabolome and allowed elucidation of the biodistribution of the endogenous metabolites. Additionally, we were able to determine the spatially integrated abundances of the ATR inhibitor ceralasertib from decontaminated human biopsies using desorption electrospray ionization-MSI (DESI-MSI).
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Descontaminación/métodos , Rayos Ultravioleta , Animales , Azetidinas/análisis , Azetidinas/uso terapéutico , COVID-19/patología , COVID-19/virología , Neoplasias de Cabeza y Cuello/química , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Metaboloma , Naftalenos/análisis , Naftalenos/uso terapéutico , Fotólisis/efectos de la radiación , Ratas , Ratas Wistar , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/efectos de la radiación , Espectrometría de Masa por Ionización de Electrospray/métodos , Terfenadina/química , Inactivación de Virus/efectos de la radiaciónRESUMEN
Imaging mass cytometry (IMC) offers the opportunity to image metal- and heavy halogen-containing xenobiotics in a highly multiplexed experiment with other immunochemistry-based reagents to distinguish uptake into different tissue structures or cell types. However, in practice, many xenobiotics are not amenable to this analysis, as any compound which is not bound to the tissue matrix will delocalize during aqueous sample-processing steps required for IMC analysis. Here, we present a strategy to perform IMC experiments on a water-soluble polysarcosine-modified dendrimer drug-delivery system (S-Dends). This strategy involves two consecutive imaging acquisitions on the same tissue section using the same instrumental platform, an initial laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MSI) experiment followed by tissue staining and a standard IMC experiment. We demonstrated that settings can be found for the initial ablation step that leave sufficient residual tissue for subsequent antibody staining and visualization. This workflow results in lateral resolution for the S-Dends of 2 µm followed by imaging of metal-tagged antibodies at 1 µm.