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
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
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
Tumor vaccine therapy offers significant advantages over conventional treatments,including reduced toxic side effects.However,it currently functions primarily as an adjuvant treatment modality in clinical oncology due...Tumor vaccine therapy offers significant advantages over conventional treatments,including reduced toxic side effects.However,it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods.Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors,thereby limiting their clinical efficacy.In this study,we developed a nanoparticle-based tumor vaccine,OVA@HA-PEI,utilizing ovalbumin(OVA)as the presenting antigen and hyaluronic acid(HA)and polyethyleneimine(PEI)as adjuvants and carriers.This formulation significantly enhanced the proliferation of immune cells and cytokines,such as CD3,CD8,interferon-,and tumor necrosis factor-,in vivo,effectively activating an immune response against B16–F10 tumors.In vivofluorescenceflow cytometry(IVFC)has already become an effective method for monitoring circulating tumor cells(CTCs)due to its direct,noninvasive,and long-term detection capabilities.Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1(PD-1)antibody.The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone.Additionally,this combination therapy substantially reduced the number of CTCs in vivo,increased the clearance rate of CTCs by the immune system,and slowed tumor progression.Thesefindings greatly enhance the clinical application prospects of IVFC and tumor vaccines.展开更多
目的慢性肾病的进展与肾脏空间免疫微环境的紊乱密切相关,本研究旨在利用成像质谱流式技术(imaging mass cytometry,IMC),开发出一套解析慢性肾病免疫微环境的实验和分析体系,以用于全景式解析慢性肾病肾脏组织的单细胞空间免疫微环境...目的慢性肾病的进展与肾脏空间免疫微环境的紊乱密切相关,本研究旨在利用成像质谱流式技术(imaging mass cytometry,IMC),开发出一套解析慢性肾病免疫微环境的实验和分析体系,以用于全景式解析慢性肾病肾脏组织的单细胞空间免疫微环境。方法(1)筛选针对肾脏结构单元、基质细胞和免疫细胞的抗原标志物,构建质谱流式成像抗体组合,金属标记的抗体均通过免疫组织化学实验完成抗体特异性验证;(2)选取2例输尿管癌旁的正常肾脏活检组织、2例糖尿病肾病(diabetic nephropathy,DN)和2例免疫球蛋白A肾病(IgA nephropathy,IgAN)患者的肾穿刺活检组织,利用上述抗体进行染色孵育,通过Hyperion成像系统完成图形数据采集;(3)整合Ilastik与CellProfiler技术建立针对肾组织的单细胞分割流程,结合手动圈门与Phenograph无监督聚类算法完成细胞身份注释;(4)建立细胞间的空间邻近关系,利用置换检验对随机分布模型进行模拟,解析组织细胞与免疫细胞之间的空间关系;(5)采用k近邻(k-nearest neighbors,kNN)算法进行空间聚类,通过细胞邻域(cellular neighborhoods,CNs)划分出肾脏组织的空间功能单元,进而分析这些功能单元的免疫学特征和比例。结果(1)构建并优化出了1个37色的抗体组合,抗体均显示出高特异性和低背景噪声,该抗体组合可精准识别肾脏的结构细胞,基质细胞和浸润的免疫细胞;(2)通过Hyperion成像系统获得正常与2种慢性肾病的肾组织高维成像图片;(3)通过单细胞拆分流程系统绘制了21种肾脏组织细胞和17种免疫细胞的人类肾脏高分辨率单细胞图谱,鉴定出了CD15^(+)巨噬细胞、GzmK^(+)CD4^(+)效应T细胞等免疫细胞以及3种新型的双阳性肾小管细胞,展现了DN与IgAN肾病组织中浸润免疫细胞的差异性;(4)建立了单细胞的空间互作分析方法,展现了慢性肾病组织中免疫细胞空间互作网络,发现肌成纤维细胞作为连接组织损伤与免疫细胞浸润的关键空间枢纽;(5)通过空间邻域分析可以将肾脏结构划分为肾小球区、近端小管区、髓袢升支区、血管和纤维化区与免疫细胞富集区等9个功能单元。病变组织中正常功能结构区域占比下降,而纤维化与免疫富集区比例显著上升。其中免疫细胞富集区普遍与各功能区域保持紧密共定位,而血管和纤维化区主要与收集管区和髓袢升支区相邻。结论本研究成功开发了一套适用于肾脏组织成像的IMC方法学体系。该体系能高分辨率全景展现免疫细胞在慢性肾脏组织中的空间分布,解析了免疫细胞与组织细胞空间互作和局部免疫微环境特征。本技术将推动基于空间特征的肾脏病精准诊断和靶向治疗策略开发。展开更多
Heavy metals currently pose one of the most serious problems in terms of environmental protection. We analysed the effect of cadmium and mercury salts upon chlorophyll fluorescence emision of Microcystis aeruginosa, C...Heavy metals currently pose one of the most serious problems in terms of environmental protection. We analysed the effect of cadmium and mercury salts upon chlorophyll fluorescence emision of Microcystis aeruginosa, Cylindrospermopsis raciborskii and Aphanizomenon gracile. Time of incubation （3, 12 and 24 h） and salts concentration（1 μM, 10 μM, 100μM） influenced the emision. The higher the salt concentration and the longer the time of incubation, the greater the influence on inhibition of chlorophyll fluorescence was observed. Our results might indicate that nontoxic strains of M. aeruginosa MAKR0205 can have a high tolerance to heavy metal ions and ability of their detoxication. Further studies are necessary to confirm their potential ability in the water purification.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 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.
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 Research and Development Program of China,Grant Number:2021YFF0502900,2019YFC1604604National Natural Science Foundation of China,Grant Number:62075013,62027824.
文摘Tumor vaccine therapy offers significant advantages over conventional treatments,including reduced toxic side effects.However,it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods.Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors,thereby limiting their clinical efficacy.In this study,we developed a nanoparticle-based tumor vaccine,OVA@HA-PEI,utilizing ovalbumin(OVA)as the presenting antigen and hyaluronic acid(HA)and polyethyleneimine(PEI)as adjuvants and carriers.This formulation significantly enhanced the proliferation of immune cells and cytokines,such as CD3,CD8,interferon-,and tumor necrosis factor-,in vivo,effectively activating an immune response against B16–F10 tumors.In vivofluorescenceflow cytometry(IVFC)has already become an effective method for monitoring circulating tumor cells(CTCs)due to its direct,noninvasive,and long-term detection capabilities.Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1(PD-1)antibody.The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone.Additionally,this combination therapy substantially reduced the number of CTCs in vivo,increased the clearance rate of CTCs by the immune system,and slowed tumor progression.Thesefindings greatly enhance the clinical application prospects of IVFC and tumor vaccines.
文摘Heavy metals currently pose one of the most serious problems in terms of environmental protection. We analysed the effect of cadmium and mercury salts upon chlorophyll fluorescence emision of Microcystis aeruginosa, Cylindrospermopsis raciborskii and Aphanizomenon gracile. Time of incubation （3, 12 and 24 h） and salts concentration（1 μM, 10 μM, 100μM） influenced the emision. The higher the salt concentration and the longer the time of incubation, the greater the influence on inhibition of chlorophyll fluorescence was observed. Our results might indicate that nontoxic strains of M. aeruginosa MAKR0205 can have a high tolerance to heavy metal ions and ability of their detoxication. Further studies are necessary to confirm their potential ability in the water purification.
