Time-resolved flow cytometry(TRFC)was used to measure metabolic differences in estrogen receptor-positive breast cancer cells.This specialty cytometry technique measures fluorescence lifetimes as a single-cell paramet...Time-resolved flow cytometry(TRFC)was used to measure metabolic differences in estrogen receptor-positive breast cancer cells.This specialty cytometry technique measures fluorescence lifetimes as a single-cell parameter thereby providing a unique approach for high-throughput cell counting and screening.Differences in fluorescence lifetime were detected and this was associated with sensitivity to the commonly prescribed therapeutic tamoxifen.Differences in fluorescence lifetime are attributed to the binding states of the autofluorescent metabolite NAD(P)H.The function of NAD(P)H is well described and in general involves cycling from a reduced to oxidized state to facilitate electron transport for the conversion of pyruvate to lactate.NAD(P)H fluorescence lifetimes depend on the bound or unbound state of the metabolite,which also relates to metabolic transitions between oxidative phosphorylation and glycolysis.To determine if fundamental metabolic profiles differ for cells that are sensitive to tamoxifen compared to those that are resistant,large populations of MCF-7 breast cancer cells were screened and fluorescence lifetimes were quantified.Additionally,metabolic differences associated with tamoxifen sensitivity were measured with a Seahorse HS mini metabolic analyzer(Agilent Technologies Inc.Santa Clara,CA)and confocal imaging.Results show that tamoxifen-resistant breast cancer cells have increased utilization of glycolysis for energy production compared to tamoxifen-sensitive breast cancer cells.This work is impacting because it establishes an early step toward developing a reliable screening technology in which large cell censuses can be differentiated for drug sensitivity in a label-free fashion.展开更多
In fluorescence flow cytometry,spectral overlap among multiple fluorescent labels cannot be avoided,and thus detected fluorescent intensities need to be compensated.Although fluorescent compensation in flow cytometry ...In fluorescence flow cytometry,spectral overlap among multiple fluorescent labels cannot be avoided,and thus detected fluorescent intensities need to be compensated.Although fluorescent compensation in flow cytometry has been widely used for many years,it still lacks quantitative evaluations to validate its effectiveness.Using a home-developed nine-color fluorescence flow cytometer,this study first obtains calibration curves by assaying gradient concentrations of nine different fluorochromes individually,with the fluorescent intensities of the highest concentrations of each fluorochrome being used to obtain a spillover matrix.Mixed fluorescent solutions are analyzed by flow cytometry in which the obtained fluorescent intensities are compensated by the spillover matrix,translated to specific concentrations based on calibration curve and compared with nominal values.Three mixed solutions of Brilliant Violet 650 and Brilliant Violet 711,of Alexa Fluor 488 and PE,and of Pacific Orange,Alexa Fluor 488,and PE are tested,with fluorescent compensation being observed to reduce excessive signals due to spectral overlap.Specifically,concentration deviations(before vs after compensation)in comparison with nominal values for Brilliant Violet 711 and Alexa Fluor 488 are quantified as 40.6%vs 14.9%and 6.7%vs 1.9%,respectively.The results presented here provide a quantitative reference for fluorescent compensation that can be used to effectively address the issue of spectral overlap in fluorescence flow cytometry.展开更多
Imaging mass cytometry(IMC)enables the high-resolution spatial profiling of tumor microenvironment,but its clinical utility for prospective prediction remains underdeveloped.In this study,we integrated IMC into a clin...Imaging mass cytometry(IMC)enables the high-resolution spatial profiling of tumor microenvironment,but its clinical utility for prospective prediction remains underdeveloped.In this study,we integrated IMC into a clinical trial of hepatocellular carcinoma(HCC)patients undergoing combination therapy with programmed death-1 blockade and transarterial chemoembolization.We analyzed 281 regions of interest from 43 patients using a custom 40-marker IMC panel and developed a novel superpixel-based graph attention network,IMCSGAT,to model spatial cell interactions within the tumor microenvironment.IMCSGAT enabled accurate multitask prediction of key clinical features,including Barcelona Clinic Liver Cancer stage,trabecular histologic subtype,and treatment response.Compared to state-of-the-art methods,IMCSGAT achieved superior performance across all classification tasks.Spatial interaction analysis revealed that resident macrophage-centered interactions,particularly those with NK and T cells,were enriched in responders and predictive of therapeutic outcome.These findings were validated in a murine HCC model,reinforcing the role of innate immune architecture in shaping the treatment response.This study establishes IMCSGAT as a powerful spatial learning framework for high-dimensional IMC data,with potential applications in clinical outcome prediction and personalized therapy design for HCC.Our results provide a blueprint for the broader use of spatial analytics in precision oncology.展开更多
Bladder cancer(BC)is a common malignancy and among the leading causes of cancer death worldwide.Analysis of BC cells is of great significance for clinical diagnosis and disease treatment.Current approaches rely mainly...Bladder cancer(BC)is a common malignancy and among the leading causes of cancer death worldwide.Analysis of BC cells is of great significance for clinical diagnosis and disease treatment.Current approaches rely mainly on imaging-based technology,which requires complex staining and sophisticated instrumentation.In this work,we develop a label-free method based on artificial intelligence(AI)-assisted impedance-based flow cytometry(IFC)to differentiate between various BC cells and epithelial cells at single-cell resolution.By applying multiple-frequency excitations,the electrical characteristics of cells,including membrane and nuclear opacities,are extracted,allowing distinction to be made between epithelial cells,low-grade,and high-grade BC cells.Through the use of a constriction channel,the electro-mechanical properties associated with active deformation behavior of cells are investigated,and it is demonstrated that BC cells have a greater capability of shape recovery,an observation that further increases differentiation accuracy.With the assistance of a convolutional neural network-based AI algorithm,IFC is able to effectively differentiate various BC and epithelial cells with accuracies of over 95%.In addition,different grades of BC cells are successfully differentiated in both spiked mixed samples and bladder tumor tissues.展开更多
Triple-negative bresst canær(TNBC)metastscis is particularly severe due to its aggressive nsture,leading to rapid disease progresion and significantly reduced survival rates.Rujifang(RJF),a traditional Chinese fo...Triple-negative bresst canær(TNBC)metastscis is particularly severe due to its aggressive nsture,leading to rapid disease progresion and significantly reduced survival rates.Rujifang(RJF),a traditional Chinese formula,has demonstrated potential anti-tumor effects and theability to inhibit TNBC metastasis.However,the efects af varying R.IF dors remain undear.This study utilized Laser-based in vino fow cytometry(IVFC)to monitor circulating tumor cells(CTCs)and evaluate the efficacy of R.IF at different doses.The results indicated that R.IF at the high dose inhibited both the number af CTC:and the formaton of metatatic foci more eflectively compared to the lower dose.TUNEL assays revealed that R.IF trentment promotes apoptosis of tumor cells,with a more pronounced effect observed at the higher dose.Immuno-fluorescence experiments demonstrated that administering a higher dose of R.IF suppreses theеxprescion of Kindlin-1 more effectively in the tumor microenvironment.Although higher doses showed enhanced efficacy,they might also lesd to an increase in side efects.These findings underscore the promise and challenges of using R.IF at high doses for anti-tumor therspy.They highlight the criticnl importance of optimizing the dose of R.JP in the treatment of TNBC and provide valuable insights for its dinical application.展开更多
Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and periphe...Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.展开更多
To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Meth...To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysisof two types of cyclins and DNA content simultaneously in individual cells by two-laser flowcytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. ATriton-X100 permeabilization procedure was optimized for detection of two types of cyclins. Onecyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other,indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochromeDAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method.Results: Permeabilization with 0.5% Triton-XlOO in PBS containing 1% BSA for 5 min on ice providedoptimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It wasfound that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured withno compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growingMOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously,allowing for analysis of cell cycle phase specific perturbations without the necessity of cellsynchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine andcyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleledexpected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only beapplied to measure unscheduled vs. expected cyclin expression but may also be used to estimate thefraction of cycling cells in up to 6 cell populations.展开更多
目的探讨外周血淋巴细胞亚群在肺动脉高压(PAH)诊断中的潜在价值。方法选取89例PAH患者与100例健康对照者,采用流式细胞术检测外周血淋巴细胞亚群(CD3+、CD4+、CD8+T细胞、NK细胞、CD19+B细胞)的百分比与绝对值,并通过ROC曲线评估其诊...目的探讨外周血淋巴细胞亚群在肺动脉高压(PAH)诊断中的潜在价值。方法选取89例PAH患者与100例健康对照者,采用流式细胞术检测外周血淋巴细胞亚群(CD3+、CD4+、CD8+T细胞、NK细胞、CD19+B细胞)的百分比与绝对值,并通过ROC曲线评估其诊断效能。结果PAH患者呈现显著的免疫失衡特征:中性粒细胞计数升高而淋巴细胞计数降低;在淋巴细胞亚群中,NK细胞的百分比与绝对值均显著降低(绝对值:107.0 vs 254.5个/μL,P<0.0001),其诊断PAH的效能最高,ROC曲线下面积(AUC)达0.8296。结论PAH患者外周血存在以NK细胞显著减少为特征的免疫紊乱,NK细胞绝对值对PAH具有良好的诊断价值,有望成为潜在的辅助诊断生物标志物。展开更多
目的·使用质谱流式细胞技术(cytometry by time-of-flight,CyTOF)分析乳腺癌患者肿瘤组织的多种抗原,探究其与乳腺癌微环境、乳腺癌患者预后的关系。方法·使用Maxpar^(■)Panel Designer v2.0.1软件结合相关抗原蛋白及组织细...目的·使用质谱流式细胞技术(cytometry by time-of-flight,CyTOF)分析乳腺癌患者肿瘤组织的多种抗原,探究其与乳腺癌微环境、乳腺癌患者预后的关系。方法·使用Maxpar^(■)Panel Designer v2.0.1软件结合相关抗原蛋白及组织细胞标志物设计Panel。使用Maxpar X8抗体标记试剂盒将相关镧系(Ln)金属同位素与Panel中的蛋白抗体连接后,采用成像质谱流式细胞染色(imaging mass cytometry staining,IMC)法对乳腺癌组织芯片进行染色。在Hyperion成像系统中观察,得到Panel中多种蛋白标志物的表达和空间定位。使用R语言对原始数据进行数据归一化、去噪和降噪、补偿校正以及数据转换,再进行降维处理。通过聚类算法进行细胞亚群注释。使用空间邻域分析,解析乳腺癌微环境中的各类细胞和临床意义。结果·将金属标签与相应的抗原抗体连接后,染色效果良好,可用于IMC染色。在乳腺癌组织芯片中,根据现有的26种标志物可以将乳腺癌微环境分成9种细胞类型,共410000个细胞。在配对的肿瘤组织和癌旁组织中,乳腺癌微环境主要由B细胞、CD4^(+)T细胞、CD8^(+)T细胞、上皮细胞、内皮细胞、巨噬细胞、肌上皮细胞、中性粒细胞、成纤维细胞组成。其中,巨噬细胞和CD4^(+)T细胞在肿瘤组织与癌旁组织中的数量差异有统计学意义(均P<0.05)。在乳腺癌组织中鉴定出15种特征性细胞邻域,其中CD8^(+)T细胞和巨噬细胞与肿瘤细胞空间共定位邻域,与患者生存期延长显著相关(P=0.011,P<0.001)。结论·CyTOF对于大批量检测多种抗原在组织中的表达有重要作用,可以在微观角度上分析乳腺癌组织与乳腺癌微环境的关系。在乳腺癌微环境中,CD8^(+)T细胞和巨噬细胞的表达量较高与乳腺癌患者的良好预后相关。展开更多
基金the National Institute of Health for supporting this research under grants NIH R35GM152076,NIH 1SC1GM127175-01,NIH T32GM148394.
文摘Time-resolved flow cytometry(TRFC)was used to measure metabolic differences in estrogen receptor-positive breast cancer cells.This specialty cytometry technique measures fluorescence lifetimes as a single-cell parameter thereby providing a unique approach for high-throughput cell counting and screening.Differences in fluorescence lifetime were detected and this was associated with sensitivity to the commonly prescribed therapeutic tamoxifen.Differences in fluorescence lifetime are attributed to the binding states of the autofluorescent metabolite NAD(P)H.The function of NAD(P)H is well described and in general involves cycling from a reduced to oxidized state to facilitate electron transport for the conversion of pyruvate to lactate.NAD(P)H fluorescence lifetimes depend on the bound or unbound state of the metabolite,which also relates to metabolic transitions between oxidative phosphorylation and glycolysis.To determine if fundamental metabolic profiles differ for cells that are sensitive to tamoxifen compared to those that are resistant,large populations of MCF-7 breast cancer cells were screened and fluorescence lifetimes were quantified.Additionally,metabolic differences associated with tamoxifen sensitivity were measured with a Seahorse HS mini metabolic analyzer(Agilent Technologies Inc.Santa Clara,CA)and confocal imaging.Results show that tamoxifen-resistant breast cancer cells have increased utilization of glycolysis for energy production compared to tamoxifen-sensitive breast cancer cells.This work is impacting because it establishes an early step toward developing a reliable screening technology in which large cell censuses can be differentiated for drug sensitivity in a label-free fashion.
基金support from the National Natural Science Foundation of China(Grant Nos.62331025 and 62121003).
文摘In fluorescence flow cytometry,spectral overlap among multiple fluorescent labels cannot be avoided,and thus detected fluorescent intensities need to be compensated.Although fluorescent compensation in flow cytometry has been widely used for many years,it still lacks quantitative evaluations to validate its effectiveness.Using a home-developed nine-color fluorescence flow cytometer,this study first obtains calibration curves by assaying gradient concentrations of nine different fluorochromes individually,with the fluorescent intensities of the highest concentrations of each fluorochrome being used to obtain a spillover matrix.Mixed fluorescent solutions are analyzed by flow cytometry in which the obtained fluorescent intensities are compensated by the spillover matrix,translated to specific concentrations based on calibration curve and compared with nominal values.Three mixed solutions of Brilliant Violet 650 and Brilliant Violet 711,of Alexa Fluor 488 and PE,and of Pacific Orange,Alexa Fluor 488,and PE are tested,with fluorescent compensation being observed to reduce excessive signals due to spectral overlap.Specifically,concentration deviations(before vs after compensation)in comparison with nominal values for Brilliant Violet 711 and Alexa Fluor 488 are quantified as 40.6%vs 14.9%and 6.7%vs 1.9%,respectively.The results presented here provide a quantitative reference for fluorescent compensation that can be used to effectively address the issue of spectral overlap in fluorescence flow cytometry.
基金supported by the National Natural Science Foundation of China(grant 62136004 to D.Z.,grant 62272226 to W.S.,and grant 82173078 to J.S.)the National Key Research and Development Program of China(grant 2019YFA0803000 to J.S.)+1 种基金the Excellent Youth Foundation of Zhejiang Scientific(grant R22H1610037 to J.S.)the Zhejiang Provincial Natural Science Foundation(grant 2022C03037 to J.S.).
文摘Imaging mass cytometry(IMC)enables the high-resolution spatial profiling of tumor microenvironment,but its clinical utility for prospective prediction remains underdeveloped.In this study,we integrated IMC into a clinical trial of hepatocellular carcinoma(HCC)patients undergoing combination therapy with programmed death-1 blockade and transarterial chemoembolization.We analyzed 281 regions of interest from 43 patients using a custom 40-marker IMC panel and developed a novel superpixel-based graph attention network,IMCSGAT,to model spatial cell interactions within the tumor microenvironment.IMCSGAT enabled accurate multitask prediction of key clinical features,including Barcelona Clinic Liver Cancer stage,trabecular histologic subtype,and treatment response.Compared to state-of-the-art methods,IMCSGAT achieved superior performance across all classification tasks.Spatial interaction analysis revealed that resident macrophage-centered interactions,particularly those with NK and T cells,were enriched in responders and predictive of therapeutic outcome.These findings were validated in a murine HCC model,reinforcing the role of innate immune architecture in shaping the treatment response.This study establishes IMCSGAT as a powerful spatial learning framework for high-dimensional IMC data,with potential applications in clinical outcome prediction and personalized therapy design for HCC.Our results provide a blueprint for the broader use of spatial analytics in precision oncology.
基金financial support from the National Natural Science Foundation of China(NSFC Grant No.22076138)the National Natural Science Foundation of China(NSFC Grant No.62174119).
文摘Bladder cancer(BC)is a common malignancy and among the leading causes of cancer death worldwide.Analysis of BC cells is of great significance for clinical diagnosis and disease treatment.Current approaches rely mainly on imaging-based technology,which requires complex staining and sophisticated instrumentation.In this work,we develop a label-free method based on artificial intelligence(AI)-assisted impedance-based flow cytometry(IFC)to differentiate between various BC cells and epithelial cells at single-cell resolution.By applying multiple-frequency excitations,the electrical characteristics of cells,including membrane and nuclear opacities,are extracted,allowing distinction to be made between epithelial cells,low-grade,and high-grade BC cells.Through the use of a constriction channel,the electro-mechanical properties associated with active deformation behavior of cells are investigated,and it is demonstrated that BC cells have a greater capability of shape recovery,an observation that further increases differentiation accuracy.With the assistance of a convolutional neural network-based AI algorithm,IFC is able to effectively differentiate various BC and epithelial cells with accuracies of over 95%.In addition,different grades of BC cells are successfully differentiated in both spiked mixed samples and bladder tumor tissues.
基金supported by the National Key Re-search and Development Program of China(2021YFF0502900,2019YFC1604604)the grant of Peak Climbing Project of Foshan Hospital of Tra-ditional Chinese Medicine,Traditional Chinese Medicine Bureat of Guangdong Province Project(No.20213018)+2 种基金the Special Fund for Research on National Major Research Instruuments of China(Grant No.62027824)Scientific Research Fund of Education Department of Yunnan Province(2023Y0619)Biomedical Projects of Yun-nan Key Science and Technology Program(202302AA310046).
文摘Triple-negative bresst canær(TNBC)metastscis is particularly severe due to its aggressive nsture,leading to rapid disease progresion and significantly reduced survival rates.Rujifang(RJF),a traditional Chinese formula,has demonstrated potential anti-tumor effects and theability to inhibit TNBC metastasis.However,the efects af varying R.IF dors remain undear.This study utilized Laser-based in vino fow cytometry(IVFC)to monitor circulating tumor cells(CTCs)and evaluate the efficacy of R.IF at different doses.The results indicated that R.IF at the high dose inhibited both the number af CTC:and the formaton of metatatic foci more eflectively compared to the lower dose.TUNEL assays revealed that R.IF trentment promotes apoptosis of tumor cells,with a more pronounced effect observed at the higher dose.Immuno-fluorescence experiments demonstrated that administering a higher dose of R.IF suppreses theеxprescion of Kindlin-1 more effectively in the tumor microenvironment.Although higher doses showed enhanced efficacy,they might also lesd to an increase in side efects.These findings underscore the promise and challenges of using R.IF at high doses for anti-tumor therspy.They highlight the criticnl importance of optimizing the dose of R.JP in the treatment of TNBC and provide valuable insights for its dinical application.
文摘Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.
基金This project was supported by grants from China Key Basic Research Program Grant (No. G1998051212) the National Natural Sciences Foundation of China (No. 39670265, 39730270 and 39725027) grants from the Science Foundation of Ministry of Public Health, China (No. 202-01-06).
文摘To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysisof two types of cyclins and DNA content simultaneously in individual cells by two-laser flowcytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. ATriton-X100 permeabilization procedure was optimized for detection of two types of cyclins. Onecyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other,indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochromeDAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method.Results: Permeabilization with 0.5% Triton-XlOO in PBS containing 1% BSA for 5 min on ice providedoptimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It wasfound that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured withno compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growingMOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously,allowing for analysis of cell cycle phase specific perturbations without the necessity of cellsynchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine andcyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleledexpected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only beapplied to measure unscheduled vs. expected cyclin expression but may also be used to estimate thefraction of cycling cells in up to 6 cell populations.
文摘目的探讨外周血淋巴细胞亚群在肺动脉高压(PAH)诊断中的潜在价值。方法选取89例PAH患者与100例健康对照者,采用流式细胞术检测外周血淋巴细胞亚群(CD3+、CD4+、CD8+T细胞、NK细胞、CD19+B细胞)的百分比与绝对值,并通过ROC曲线评估其诊断效能。结果PAH患者呈现显著的免疫失衡特征:中性粒细胞计数升高而淋巴细胞计数降低;在淋巴细胞亚群中,NK细胞的百分比与绝对值均显著降低(绝对值:107.0 vs 254.5个/μL,P<0.0001),其诊断PAH的效能最高,ROC曲线下面积(AUC)达0.8296。结论PAH患者外周血存在以NK细胞显著减少为特征的免疫紊乱,NK细胞绝对值对PAH具有良好的诊断价值,有望成为潜在的辅助诊断生物标志物。
文摘目的·使用质谱流式细胞技术(cytometry by time-of-flight,CyTOF)分析乳腺癌患者肿瘤组织的多种抗原,探究其与乳腺癌微环境、乳腺癌患者预后的关系。方法·使用Maxpar^(■)Panel Designer v2.0.1软件结合相关抗原蛋白及组织细胞标志物设计Panel。使用Maxpar X8抗体标记试剂盒将相关镧系(Ln)金属同位素与Panel中的蛋白抗体连接后,采用成像质谱流式细胞染色(imaging mass cytometry staining,IMC)法对乳腺癌组织芯片进行染色。在Hyperion成像系统中观察,得到Panel中多种蛋白标志物的表达和空间定位。使用R语言对原始数据进行数据归一化、去噪和降噪、补偿校正以及数据转换,再进行降维处理。通过聚类算法进行细胞亚群注释。使用空间邻域分析,解析乳腺癌微环境中的各类细胞和临床意义。结果·将金属标签与相应的抗原抗体连接后,染色效果良好,可用于IMC染色。在乳腺癌组织芯片中,根据现有的26种标志物可以将乳腺癌微环境分成9种细胞类型,共410000个细胞。在配对的肿瘤组织和癌旁组织中,乳腺癌微环境主要由B细胞、CD4^(+)T细胞、CD8^(+)T细胞、上皮细胞、内皮细胞、巨噬细胞、肌上皮细胞、中性粒细胞、成纤维细胞组成。其中,巨噬细胞和CD4^(+)T细胞在肿瘤组织与癌旁组织中的数量差异有统计学意义(均P<0.05)。在乳腺癌组织中鉴定出15种特征性细胞邻域,其中CD8^(+)T细胞和巨噬细胞与肿瘤细胞空间共定位邻域,与患者生存期延长显著相关(P=0.011,P<0.001)。结论·CyTOF对于大批量检测多种抗原在组织中的表达有重要作用,可以在微观角度上分析乳腺癌组织与乳腺癌微环境的关系。在乳腺癌微环境中,CD8^(+)T细胞和巨噬细胞的表达量较高与乳腺癌患者的良好预后相关。
