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Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.
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Anticorpos , Recursos Comunitários , Humanos , Reprodutibilidade dos Testes , Diagnóstico por ImagemRESUMO
Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.
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Anticorpos , Comunicação Celular , Diagnóstico por ImagemRESUMO
Colorectal cancer (CRC) is one of the most frequently occurring cancers, but prognostic biomarkers identifying patients at risk of recurrence are still lacking. In this study, we aimed to investigate in more detail the spatial relationship between intratumoural T cells, cancer cells, and cancer cell hallmarks as prognostic biomarkers in stage III colorectal cancer patients. We conducted multiplexed imaging of 56 protein markers at single-cell resolution on resected fixed tissue from stage III CRC patients who received adjuvant 5-fluorouracil (5FU)-based chemotherapy. Images underwent segmentation for tumour, stroma, and immune cells, and cancer cell 'state' protein marker expression was quantified at a cellular level. We developed a Python package for estimation of spatial proximity, nearest neighbour analysis focusing on cancer cell-T-cell interactions at single-cell level. In our discovery cohort (Memorial Sloan Kettering samples), we processed 462 core samples (total number of cells: 1,669,228) from 221 adjuvant 5FU-treated stage III patients. The validation cohort (Huntsville Clearview Cancer Center samples) consisted of 272 samples (total number of cells: 853,398) from 98 stage III CRC patients. While there were trends for an association between the percentage of cytotoxic T cells (across the whole cancer core), it did not reach significance (discovery cohort: p = 0.07; validation cohort: p = 0.19). We next utilised our region-based nearest neighbour approach to determine the spatial relationships between cytotoxic T cells, helper T cells, and cancer cell clusters. In both cohorts, we found that shorter distance between cytotoxic T cells, T helper cells, and cancer cells was significantly associated with increased disease-free survival. An unsupervised trained model that clustered patients based on the median distance between immune cells and cancer cells, as well as protein expression profiles, successfully classified patients into low-risk and high-risk groups (discovery cohort: p = 0.01; validation cohort: p = 0.003). © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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BACKGROUND: Existing colorectal cancer subtyping methods were generated without much consideration of potential differences in expression profiles between colon and rectal tissues. Moreover, locally advanced rectal cancers at resection often have received neoadjuvant chemoradiotherapy which likely has a significant impact on gene expression. METHODS: We collected mRNA expression profiles for rectal and colon cancer samples (n = 2121). We observed that (i) Consensus Molecular Subtyping (CMS) had a different prognosis in treatment-naïve rectal vs. colon cancers, and (ii) that neoadjuvant chemoradiotherapy exposure produced a strong shift in CMS subtypes in rectal cancers. We therefore clustered 182 untreated rectal cancers to find rectal cancer-specific subtypes (RSSs). RESULTS: We identified three robust subtypes. We observed that RSS1 had better, and RSS2 had worse disease-free survival. RSS1 showed high expression of MYC target genes and low activity of angiogenesis genes. RSS2 exhibited low regulatory T cell abundance, strong EMT and angiogenesis signalling, and high activation of TGF-ß, NF-κB, and TNF-α signalling. RSS3 was characterised by the deactivation of EGFR, MAPK and WNT pathways. CONCLUSIONS: We conclude that RSS subtyping allows for more accurate prognosis predictions in rectal cancers than CMS subtyping and provides new insight into targetable disease pathways within these subtypes.
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Neoplasias Retais , Humanos , Neoplasias Retais/genética , Neoplasias Retais/patologia , Neoplasias Retais/terapia , Neoplasias Retais/classificação , Prognóstico , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/classificação , Perfilação da Expressão Gênica , Terapia NeoadjuvanteRESUMO
BACKGROUND: Loss of AZGP1 expression is a biomarker associated with progression to castration resistance, development of metastasis, and poor disease-specific survival in prostate cancer. However, high expression of AZGP1 cells in prostate cancer has been reported to increase proliferation and invasion. The exact role of AZGP1 in prostate cancer progression remains elusive. METHOD: AZGP1 knockout and overexpressing prostate cancer cells were generated using a lentiviral system. The effects of AZGP1 under- or over-expression in prostate cancer cells were evaluated by in vitro cell proliferation, migration, and invasion assays. Heterozygous AZGP1± mice were obtained from European Mouse Mutant Archive (EMMA), and prostate tissues from homozygous knockout male mice were collected at 2, 6 and 10 months for histological analysis. In vivo xenografts generated from AZGP1 under- or over-expressing prostate cancer cells were used to determine the role of AZGP1 in prostate cancer tumor growth, and subsequent proteomics analysis was conducted to elucidate the mechanisms of AZGP1 action in prostate cancer progression. AZGP1 expression and microvessel density were measured in human prostate cancer samples on a tissue microarray of 215 independent patient samples. RESULT: Neither the knockout nor overexpression of AZGP1 exhibited significant effects on prostate cancer cell proliferation, clonal growth, migration, or invasion in vitro. The prostates of AZGP1-/- mice initially appeared to have grossly normal morphology; however, we observed fibrosis in the periglandular stroma and higher blood vessel density in the mouse prostate by 6 months. In PC3 and DU145 mouse xenografts, over-expression of AZGP1 did not affect tumor growth. Instead, these tumors displayed decreased microvessel density compared to xenografts derived from PC3 and DU145 control cells, suggesting that AZGP1 functions to inhibit angiogenesis in prostate cancer. Proteomics profiling further indicated that, compared to control xenografts, AZGP1 overexpressing PC3 xenografts are enriched with angiogenesis pathway proteins, including YWHAZ, EPHA2, SERPINE1, and PDCD6, MMP9, GPX1, HSPB1, COL18A1, RNH1, and ANXA1. In vitro functional studies show that AZGP1 inhibits human umbilical vein endothelial cell proliferation, migration, tubular formation and branching. Additionally, tumor microarray analysis shows that AZGP1 expression is negatively correlated with blood vessel density in human prostate cancer tissues. CONCLUSION: AZGP1 is a negative regulator of angiogenesis, such that loss of AZGP1 promotes angiogenesis in prostate cancer. AZGP1 likely exerts heterotypical effects on cells in the tumor microenvironment, such as stromal and endothelial cells. This study sheds light on the anti-angiogenic characteristics of AZGP1 in the prostate and provides a rationale to target AZGP1 to inhibit prostate cancer progression.
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Movimento Celular , Proliferação de Células , Neovascularização Patológica , Neoplasias da Próstata , Masculino , Animais , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Linhagem Celular Tumoral , Camundongos Knockout , Glicoproteínas/metabolismo , Invasividade Neoplásica , Camundongos , Regulação Neoplásica da Expressão Gênica , Angiogênese , Glicoproteína Zn-alfa-2RESUMO
MOTIVATION: Multiplexed immunofluorescence bioimaging of single-cells and their spatial organization in tissue holds great promise to the development of future precision diagnostics and therapeutics. Current multiplexing pipelines typically involve multiple rounds of immunofluorescence staining across multiple tissue slides. This introduces experimental batch effects that can hide underlying biological signal. It is important to have robust algorithms that can correct for the batch effects while not introducing biases into the data. Performance of data normalization methods can vary among different assay pipelines. To evaluate differences, it is critical to have a ground truth dataset that is representative of the assay. RESULTS: A new immunoFLuorescence Image NOrmalization method is presented and evaluated against alternative methods and workflows. Multiround immunofluorescence staining of the same tissue with the nuclear dye DAPI was used to represent virtual slides and a ground truth. DAPI was restained on a given tissue slide producing multiple images of the same underlying structure but undergoing multiple representative tissue handling steps. This ground truth dataset was used to evaluate and compare multiple normalization methods including median, quantile, smooth quantile, median ratio normalization and trimmed mean of the M-values. These methods were applied in both an unbiased grid object and segmented cell object workflow to 24 multiplexed biomarkers. An upper quartile normalization of grid objects in log space was found to obtain almost equivalent performance to directly normalizing segmented cell objects by the middle quantile. The developed grid-based technique was then applied with on-slide controls for evaluation. Using five or fewer controls per slide can introduce biases into the data. Ten or more on-slide controls were able to robustly correct for batch effects. AVAILABILITY AND IMPLEMENTATION: The data underlying this article along with the FLINO R-scripts used to perform the evaluation of image normalizations methods and workflows can be downloaded from https://github.com/GE-Bio/FLINO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Algoritmos , Núcleo Celular , Viés , ImunofluorescênciaRESUMO
BACKGROUND: Mucinous rectal cancer is associated with a higher incidence of microsatellite instability and a poorer response to neoadjuvant chemoradiotherapy compared to other subtypes of rectal adenocarcinoma. Immune checkpoint inhibitors are an emerging family of anticancer therapeutics associated with highly variable outcomes in colorectal cancer. Although the immune landscape of mucinous rectal cancer has not been fully explored, the presence of mucin is thought to act as a barrier preventing immune-cell infiltration. OBJECTIVE: The aim of this study was to determine the immune properties of mucinous rectal cancer and investigate the degree of lymphocyte infiltration in this cohort. DESIGN: This is a retrospective cohort study that involved multiplexed immunofluorescence staining of tumor microarrays. SETTINGS: Samples originated from a single university teaching hospital. PATIENTS: Our cohort included 15 cases of mucinous and 43 cases of nonmucinous rectal cancer. MAIN OUTCOME MEASURES: Immune cells were classified and quantified. Immune-cell counts were compared between mucinous and nonmucinous cohorts. Immune marker expression within tumor epithelial tissue was evaluated to determine the degree of lymphocyte infiltration. RESULTS: Cytotoxic ( p = 0.022) and regulatory T cells ( p = 0.010) were found to be overrepresented in the mucinous cohort compared to the nonmucinous group. Programmed cell death protein 1 expression was also found to be significantly greater in the mucinous group ( p = 0.001). CD3 ( p = 0.001) and CD8 ( p = 0.054) expressions within the tumor epithelium were also higher in the mucinous group, suggesting adequate immune infiltration despite the presence of mucin. In our analysis, microsatellite instability status was not a predictor of immune marker expression. LIMITATIONS: The relatively small size of the cohort. CONCLUSIONS: Mucinous rectal cancer is associated with an immune-rich tumor microenvironment, which was not associated with microsatellite instability status. See Video Abstract at http://links.lww.com/DCR/C65 . IMGENES DE INMUNOFLUORESCENCIA MULTIPLEXADAS REVELAN UN MICROAMBIENTE TUMORAL RICO EN INMUNIDAD EN EL CNCER RECTAL MUCINOSO CARACTERIZADO POR UNA MAYOR INFILTRACIN DE LINFOCITOS Y UNA EXPRESIN MEJORADA DE PD: ANTECEDENTES:El cáncer rectal mucinoso se asocia con una mayor incidencia de inestabilidad de microsatélites y una peor respuesta a la quimiorradioterapia neoadyuvante en comparación con otros subtipos de adenocarcinoma rectal. Los inhibidores de puntos de control inmunitarios son una familia emergente de tratamientos contra el cáncer asociados con resultados muy variables en el cáncer colorrectal. Aunque el panorama inmunitario del cáncer rectal mucinoso no se ha explorado completamente, se cree que la presencia de mucina actúa como una barrera que previene la infiltración de células inmunitarias.OBJETIVO:El objetivo de este estudio fue determinar las propiedades inmunes del cáncer de recto mucinoso e investigar el grado de infiltración de linfocitos en esta cohorte.DISEÑO:Este es un estudio de cohorte retrospectivo que involucró la tinción de inmunofluorescencia multiplexada de micromatrices tumorales.AJUSTES:Las muestras se originaron en un solo hospital docente universitario.PACIENTES:Nuestra cohorte incluyó 15 casos de cáncer de recto mucinoso y 43 casos de cáncer de recto no mucinosoPRINCIPALES MEDIDAS DE RESULTADO:Las células inmunitarias se clasificaron y cuantificaron. Se compararon los recuentos de células inmunitarias entre cohortes mucinosas y no mucinosas. Se evaluó la expresión del marcador inmunitario dentro del tejido epitelial tumoral para determinar el grado de infiltración de linfocitos.RESULTADOS:Se encontró que las células T citotóxicas ( p = 0,022) y reguladoras ( p = 0,010) estaban sobrerrepresentadas en la cohorte mucinosa en comparación con el grupo no mucinoso. También se encontró que la expresión de PD-1 era significativamente mayor en el grupo mucinoso ( p = 0,001). La expresión de CD3 ( p = 0,001) y CD8 ( p = 0,054) dentro del epitelio tumoral también fue mayor en el grupo mucinoso, lo que sugiere una infiltración inmunitaria adecuada a pesar de la presencia de mucina. En nuestro análisis, no se encontró que el estado de inestabilidad de los microsatélites sea un predictor de la expresión del marcador inmunitario.LIMITACIONES:El tamaño relativamente pequeño de la cohorte.CONCLUSIONES:El cáncer rectal mucinoso se asocia con un microambiente tumoral rico en inmunidad, que no se asoció con el estado de inestabilidad de microsatélites. Consulte el Video del Resumen en http://links.lww.com/DCR/C65 . (Traducción- Dr. Yesenia Rojas-Khalil ).
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Adenocarcinoma , Neoplasias Retais , Humanos , Prognóstico , Receptor de Morte Celular Programada 1 , Estudos Retrospectivos , Microambiente Tumoral , Instabilidade de Microssatélites , Neoplasias Retais/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linfócitos/patologia , Mucinas/genética , Imunofluorescência , Terapia Neoadjuvante/métodos , Estadiamento de Neoplasias , Quimiorradioterapia/métodosRESUMO
Ylidenenorbornadienes (YNDs), prepared by [4 + 2] cycloadditions between fulvenes and acetylene carboxylates, react with thiol nucleophiles to yield mixtures of four to eight diastereomers depending on the symmetry of the YND substrate. The mixtures of diastereomers fragment via a retro-[4 + 2] cycloaddition with a large variation in rate, with half-lives ranging from 16 to 11,000 min at 80 °C. The diastereomer-enriched samples of propane thiol adducts [YND-propanethiol (PTs)] were isolated and identified by nuclear Overhauser effect spectroscopy (NOESY) correlations. Simulated kinetics were used to extrapolate the rate constants of individual diastereomers from the observed rate data, and it correlated well with rate constants measured directly and from isolated diastereomer-enriched samples. The individual diastereomers of a model system fragment at differing rates with half-lives ranging from 5 to 44 min in CDCl3. Density functional theory calculations were performed to investigate the mechanism of fragmentation and support an asynchronous retro-[4 + 2] cycloaddition transition state. The computations generally correlated well with the observed free energies of activation for four diastereomers of the model system as a whole, within 2.6 kcal/mol. However, the observed order of the fragmentation rates across the set of diastereomers deviated from the computational results. YNDs display wide variability in the rate of fragmentation, dependent on the stereoelectronics of the ylidene substituents. A Hammett study showed that the electron-rich aromatic rings attached to the ylidene bridge increase the fragmentation rate, while electron-deficient systems slow fragmentation rates.
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Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Biomarkers for identification of patients at increased risk of disease recurrence following adjuvant chemotherapy are currently lacking. In this study, we assessed immune signatures in the tumor and tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single-cell analysis technology (Cell DIVETM) and evaluated their correlations with patient outcomes. Tissue microarrays (TMAs) with up to three 1 mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin (FOLFOX) chemotherapy. Single sections underwent multiplexed immunofluorescence staining for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and tumor/cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase, and S6). We used annotations and a probabilistic classification algorithm to build statistical models of immune cell types. Images were also qualitatively assessed independently by a Pathologist as 'high', 'moderate' or 'low', for stromal and total immune cell content. Excellent agreement was found between manual assessment and total automated scores (p < 0.0001). Moreover, compared to single markers, a multi-marker classification of regulatory T cells (Tregs: CD3+/CD4+FOXP3+/PD1-) was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.049 and 0.032) of FOLFOX-treated patients. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. These findings were supported by results from an independent FOLFOX-treated cohort of 191 stage III CRC patients, where higher PD1- Tregs were associated with an increase overall survival (p = 0.015) for CD3+/CD4+/FOXP3+/PD1-. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cell types with stronger correlations with outcomes.
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Neoplasias Colorretais , Análise de Célula Única , Subpopulações de Linfócitos T , Biomarcadores Tumorais , Quimioterapia Adjuvante , Neoplasias Colorretais/patologia , Humanos , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Prognóstico , Subpopulações de Linfócitos T/citologia , Microambiente TumoralRESUMO
BACKGROUND: There is limited knowledge about DCIS cellular composition and relationship with breast cancer events (BCE). METHODS: Immunofluorescence multiplexing (MxIF) was used to image and quantify 32 cellular biomarkers in FFPE DCIS tissue microarrays. Over 75,000 DCIS cells from 51 patients (median 9 years follow-up for non-BCE cases) were analysed for profiles predictive of BCE. K-means clustering was used to evaluate cellular co-expression of epithelial markers with ER and HER2. RESULTS: Only ER, PR and HER2 significantly correlated with BCE. Cluster analysis identified 6 distinct cell groups with different levels of ER, Her2, cMET and SLC7A5. Clusters 1 and 3 were not significant. Clusters 2 and 4 (high ER/low HER2 and SLC7A5/mixed cMET) significantly correlated with low BCE risk (P = 0.001 and P = 0.034), while cluster 6 (high HER2/low ER, cMET and SLC7A5) correlated with increased risk (P = 0.018). Cluster 5 (similar to cluster 6, except high SLC7A5) trended towards significance (P = 0.072). A continuous expression score (Escore) based on these 4 clusters predicted likelihood of BCE (AUC = 0.79, log-rank test P = 5E-05; LOOCV AUC = 0.74, log-rank test P = 0.006). CONCLUSION: Multiplexed spatial analysis of limited tissue is a novel method for biomarker analysis and predicting BCEs. Further validation of Escore is needed in a larger cohort.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Mastectomia/métodos , Idoso , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/terapia , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/terapia , Terapia Combinada , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.
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Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias do Colo/diagnóstico , Formaldeído , Microscopia de Fluorescência/métodos , Inclusão em Parafina/métodos , 3,3'-Diaminobenzidina/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Receptor ErbB-2/metabolismo , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Estatísticas não Paramétricas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Patients suffering from ulcerative colitis (UC) exhibit chronic colonic inflammation caused by a dysregulated mucosal immune response and epithelial barrier disruption. Th2 cytokines, including IL-13, have been implicated in the pathogenesis of UC. IL-13 induces phosphorylation of STAT6, and we previously demonstrated increased epithelial p-STAT6 in children with UC. In this study, we investigated the role of STAT6 in oxazolone colitis, a murine model of UC, by inducing colitis in STAT6-deficient (STAT6(-/-)) and wild type (WT) mice. We observed increased epithelial cell, T cell, macrophage, and NKT cell STAT6 phosphorylation, as well as increased p-STAT6(+) IL-13-producing NKT cells, in colitic WT mice. Colitis was attenuated in STAT6(-/-) mice, with improvements in weight, colon length, and histopathology. There was decreased induction of the pore-forming tight junction protein claudin-2 in STAT6(-/-) mice. Similarly, short hairpin RNA STAT6 knockdown reduced claudin-2 induction and transepithelial resistance decrease in IL-13-treated human T84 cells. Tissue expression of IL-13, IFN-γ, IL-17, and IL-10 mRNA was similarly induced in WT and STAT6(-/-) colitic mice; however, we observed increased mRNA expression for the Th2-inducing cytokines IL-33 and thymic stromal lymphopoietin in WT mice with colitis, which was abrogated in STAT6(-/-) mice. Mesenteric lymph node cells from STAT6(-/-) mice with colitis exhibited reduced secretion of IL-4, IL-5, IL-13, and IFN-γ. IL-33 augmented mesenteric lymph node cell secretion of IL-5, IL-13, IL-6, and IFN-γ. These data implicate STAT6 in the pathogenesis of colitis in vivo with important roles in altering epithelial barrier function and regulating Th2-inducing cytokine production.
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Claudina-2/antagonistas & inibidores , Colite Ulcerativa/imunologia , Citocinas/antagonistas & inibidores , Regulação para Baixo/imunologia , Oxazolona/administração & dosagem , Fator de Transcrição STAT6/deficiência , Índice de Gravidade de Doença , Células Th2/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/antagonistas & inibidores , Animais , Linhagem Celular , Claudina-2/biossíntese , Claudina-2/genética , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/prevenção & controle , Citocinas/biossíntese , Citocinas/genética , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação da Expressão Gênica/imunologia , Haptenos/administração & dosagem , Haptenos/efeitos adversos , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/patologia , Oxazolona/efeitos adversos , Oxazolona/antagonistas & inibidores , Fator de Transcrição STAT6/genética , Células Th2/metabolismo , Células Th2/patologiaRESUMO
OBJECTIVE: The myeloid translocation genes (MTGs) are transcriptional corepressors with both Mtg8(-/-) and Mtgr1(-/-) mice showing developmental and/or differentiation defects in the intestine. We sought to determine the role of MTG16 in intestinal integrity. METHODS: Baseline and stress induced colonic phenotypes were examined in Mtg16(-/-) mice. To unmask phenotypes, we treated Mtg16(-/-) mice with dextran sodium sulphate (DSS) or infected them with Citrobacter rodentium and the colons were examined for ulceration and for changes in proliferation, apoptosis and inflammation. RESULTS: Mtg16(-/-) mice have altered immune subsets, suggesting priming towards Th1 responses. Mtg16(-/-) mice developed increased weight loss, diarrhoea, mortality and histological colitis and there were increased innate (Gr1(+), F4/80(+), CD11c(+) and MHCII(+); CD11c(+)) and Th1 adaptive (CD4) immune cells in Mtg16(-/-) colons after DSS treatment. Additionally, there was increased apoptosis and a compensatory increased proliferation in Mtg16(-/-) colons. Compared with wild-type mice, Mtg16(-/-) mice exhibited increased colonic CD4;IFN-γ cells in vehicle-treated and DSS-treated mice. Adoptive transfer of wild-type marrow into Mtg16(-/-) recipients did not rescue the Mtg16(-/-) injury phenotype. Isolated colonic epithelial cells from DSS-treated Mtg16(-/-) mice exhibited increased KC (Cxcl1) mRNA expression when compared with wild-type mice. Mtg16(-/-) mice infected with C rodentium had more severe colitis and greater bacterial colonisation. Last, MTG16 mRNA levels were reduced in human ulcerative colitis versus normal colon tissues. CONCLUSIONS: These observations indicate that MTG16 is critical for colonocyte survival and regeneration in response to intestinal injury and provide evidence that this transcriptional corepressor regulates inflammatory recruitment in response to injury.
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Colite/patologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Imunidade Adaptativa , Transferência Adotiva , Animais , Transplante Ósseo , Proliferação de Células , Colite/induzido quimicamente , Colite/imunologia , Colite/fisiopatologia , Colite Ulcerativa/metabolismo , Colo/imunologia , Sulfato de Dextrana , Enterócitos/patologia , Feminino , Humanos , Imunidade Inata , Imunofenotipagem , Absorção Intestinal/fisiologia , Mucosa Intestinal/patologia , Mucosa Intestinal/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Permeabilidade , Proteínas Repressoras , Células Th1/imunologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Colorectal cancer (CRC) is one of the most frequently occurring cancers, but prognostic biomarkers identifying patients at risk of recurrence are still lacking. In this study, we aimed to investigate in more detail the spatial relationship between intratumoural T cells, cancer cells, and cancer cell hallmarks, as prognostic biomarkers in stage III colorectal cancer patients. We conducted multiplexed imaging of 56 protein markers at single cell resolution on resected fixed tissue from stage III CRC patients who received adjuvant 5-fluorouracil-based chemotherapy. Images underwent segmentation for tumour, stroma and immune cells, and cancer cell 'state' protein marker expression was quantified at a cellular level. We developed a Python package for estimation of spatial proximity, nearest neighbour analysis focusing on cancer cell - T cell interactions at single-cell level. In our discovery cohort (MSK), we processed 462 core samples (total number of cells: 1,669,228) from 221 adjuvant 5FU-treated stage III patients. The validation cohort (HV) consisted of 272 samples (total number of cells: 853,398) from 98 stage III CRC patients. While there were trends for an association between percentage of cytotoxic T cells (across the whole cancer core), it did not reach significance (Discovery cohort: p = 0.07, Validation cohort: p = 0.19). We next utilized our region-based nearest neighbourhood approach to determine the spatial relationships between cytotoxic T cells, helper T cells and cancer cell clusters. In the both cohorts, we found that lower distance between cytotoxic T cells, T helper cells and cancer cells was significantly associated with increased disease-free survival. An unsupervised trained model that clustered patients based on the median distance between immune cells and cancer cells, as well as protein expression profiles, successfully classified patients into low-risk and high-risk groups (Discovery cohort: p = 0.01, Validation cohort: p = 0.003).
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In this novel large-scale multiplexed immunofluorescence study we comprehensively characterized and compared layer-specific proteomic features within regions of interest of the widely divergent dorsolateral prefrontal cortex (A46) and primary visual cortex (A17) of adult rhesus monkeys. Twenty-eight markers were imaged in rounds of sequential staining, and their spatial distribution precisely quantified within gray matter layers and superficial white matter. Cells were classified as neurons, astrocytes, oligodendrocytes, microglia, or endothelial cells. The distribution of fibers and blood vessels were assessed by quantification of staining intensity across regions of interest. This method revealed multivariate similarities and differences between layers and areas. Protein expression in neurons was the strongest determinant of both laminar and regional differences, whereas protein expression in glia was more important for intra-areal laminar distinctions. Among specific results, we observed a lower glia-to-neuron ratio in A17 than in A46 and the pan-neuronal markers HuD and NeuN were differentially distributed in both brain areas with a lower intensity of NeuN in layers 4 and 5 of A17 compared to A46 and other A17 layers. Astrocytes and oligodendrocytes exhibited distinct marker-specific laminar distributions that differed between regions; notably, there was a high proportion of ALDH1L1-expressing astrocytes and of oligodendrocyte markers in layer 4 of A17. The many nuanced differences in protein expression between layers and regions observed here highlight the need for direct assessment of proteins, in addition to RNA expression, and set the stage for future protein-focused studies of these and other brain regions in normal and pathological conditions.
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Mapping the human body at single cell resolution in three dimensions (3D) is important for understanding cellular interactions in context of tissue and organ organization. 2D spatial cell analysis in a single tissue section may be limited by cell numbers and histology. Here we show a workflow for 3D reconstruction of multiplexed sequential tissue sections: MATRICS-A (Multiplexed Image Three-D Reconstruction and Integrated Cell Spatial - Analysis). We demonstrate MATRICS-A in 26 serial sections of fixed skin (stained with 18 biomarkers) from 12 donors aged between 32-72 years. Comparing the 3D reconstructed cellular data with the 2D data, we show significantly shorter distances between immune cells and vascular endothelial cells (56 µm in 3D vs 108 µm in 2D). We also show 10-70% more T cells (total) within 30 µm of a neighboring T helper cell in 3D vs 2D. Distances of p53, DDB2 and Ki67 positive cells to the skin surface were consistent across all ages/sun exposure and largely localized to the lower stratum basale layer of the epidermis. MATRICS-A provides a framework for analysis of 3D spatial cell relationships in healthy and aging organs and could be further extended to diseased organs.
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Células Endoteliais , Imageamento Tridimensional , Humanos , Adulto , Pessoa de Meia-Idade , Idoso , Imageamento Tridimensional/métodos , Densidade Microvascular , Luz Solar , Envelhecimento , Contagem de CélulasRESUMO
Standard clinicopathological parameters (age, growth pattern, tumor size, margin status, and grade) have been shown to have limited value in predicting recurrence in ductal carcinoma in situ (DCIS) patients. Early and accurate recurrence prediction would facilitate a more aggressive treatment policy for high-risk patients (mastectomy or adjuvant radiation therapy), and simultaneously reduce over-treatment of low-risk patients. Generative adversarial networks (GAN) are a class of DL models in which two adversarial neural networks, generator and discriminator, compete with each other to generate high quality images. In this work, we have developed a deep learning (DL) classification network that predicts breast cancer events (BCEs) in DCIS patients using hematoxylin and eosin (H & E) images. The DL classification model was trained on 67 patients using image patches from the actual DCIS cores and GAN generated image patches to predict breast cancer events (BCEs). The hold-out validation dataset (n = 66) had an AUC of 0.82. Bayesian analysis further confirmed the independence of the model from classical clinicopathological parameters. DL models of H & E images may be used as a risk stratification strategy for DCIS patients to personalize therapy.
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Introduction: Prostate cancer is a highly heterogeneous disease, presenting varying levels of aggressiveness and response to treatment. Angiogenesis is one of the hallmarks of cancer, providing oxygen and nutrient supply to tumors. Micro vessel density has previously been correlated with higher Gleason score and poor prognosis. Manual segmentation of blood vessels (BVs) In microscopy images is challenging, time consuming and may be prone to inter-rater variabilities. In this study, an automated pipeline is presented for BV detection and distribution analysis in multiplexed prostate cancer images. Methods: A deep learning model was trained to segment BVs by combining CD31, CD34 and collagen IV images. In addition, the trained model was used to analyze the size and distribution patterns of BVs in relation to disease progression in a cohort of prostate cancer patients (N = 215). Results: The model was capable of accurately detecting and segmenting BVs, as compared to ground truth annotations provided by two reviewers. The precision (P), recall (R) and dice similarity coefficient (DSC) were equal to 0.93 (SD 0.04), 0.97 (SD 0.02) and 0.71 (SD 0.07) with respect to reviewer 1, and 0.95 (SD 0.05), 0.94 (SD 0.07) and 0.70 (SD 0.08) with respect to reviewer 2, respectively. BV count was significantly associated with 5-year recurrence (adjusted p = 0.0042), while both count and area of blood vessel were significantly associated with Gleason grade (adjusted p = 0.032 and 0.003 respectively). Discussion: The proposed methodology is anticipated to streamline and standardize BV analysis, offering additional insights into the biology of prostate cancer, with broad applicability to other cancers.
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Background: Mucinous colorectal cancer (CRC) represents 10% of all CRC and is associated with chemotherapy resistance. This study aimed to determine expression of apoptosis and necroptosis mediators in mucinous CRC. Methods: RNA gene expression data were extracted from TCGA. Protein levels in 14 mucinous and 39 non-mucinous tumors were measured by multiplexed immunofluorescence. Levels of apoptosis and necroptosis signalling proteins were analysed in SW1463 (mucinous rectal), SW837 (non-mucinous rectal), LS174T (mucinous colon) and HCT116 (non-mucinous colon) cell lines by western blot. Cell death was investigated by flow cytometry measurement of propidium iodide stained cells. Results: High cleaved-Caspase 3 expression was noted in resected mucinous tumors. Western blot identified alterations in apoptosis proteins in mucinous CRC, most prominently downregulation of Bcl-xL protein levels (p=0.029) which was also observed at the mRNA level in patients by analysis of TCGA gene expression data (p<0.001). Treatment with 5-FU did not significantly elevate cell death in mucinous cells, while non-mucinous cells showed robust cell death responses. However, 5-FU-induced phosphorylation of MLKL in mucinous cancer cells, suggestive of a switch to necroptotic cell death signaling. Conclusion: Apoptotic and necroptotic mediators are differentially expressed in mucinous and non-mucinous colorectal cancers and represent targets for investigation of cell death mechanisms in the mucinous subtype.
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Ylidenenorbornadienes (YNDs), prepared by [4 + 2] cycloadditions between fulvenes and acetylene carboxylates, react with beta-mercaptoethanol to yield a mixture of four diastereomers. These four diastereomers fragment via a retro-[4 + 2] cycloaddition at differing rates. A simulated kinetics approach extrapolated the rate constants of the diastereomers from the observed rate data. YNDs display wide variability in rate of fragmentation, dependent on the stereoelectronics of the ylidene substituents. A substrate containing one carboxylic ester proved exceptionally stable to fragmentation.