This article describes a facile approach to sort millions of cells based on their migration speed... more This article describes a facile approach to sort millions of cells based on their migration speeds in 2D or 3D microenvironments. We validate the method using imaging, transcriptomics, and functional assays for cancer and immune cells. The tool is valuable to study new correlative and causative phenotypes associated with cell migration.
ABSTRACTWhereas phenotypic assays such as Boyden chambers and wound healing assays can easily be ... more ABSTRACTWhereas phenotypic assays such as Boyden chambers and wound healing assays can easily be employed to characterize the migratory potential of cells at the population level, few methods exist that can sort subpopulations of cells based on their migratory behaviour from an initial heterogeneous pool. In this paper, we present an approach to sort migratory cancer and immune cells based on their spontaneous migration in 2D and 3D microenvironments. Using this method, which is easy to implement and readily scalable, millions of live cells can be sorted based on their migratory characteristics and then subjected to downstream genomic, molecular and functional tests. We reveal that enrichment of the most migratory cytotoxic T lymphocytes yields a pool of cells with enhanced cytotoxicity against cancer cells. This new functional sorting method opens new avenues for the precise characterization of the mechanisms underlying hitherto unexplained heterogeneities in migratory phenotypes w...
Epithelial-mesenchymal transition (EMT) has long been associated with cancer progression and meta... more Epithelial-mesenchymal transition (EMT) has long been associated with cancer progression and metastasis. As a reversible process, it has the potential to be a target for cancer therapy. However, this concept of reversing EMT has not yet been widely explored in current treatment strategies. With the identification of Nintedanib as an EMT reversal agent, we aim to broaden the utility of this triple angiokinase inhibitor by identifying agents that show synergistic lethality with it, using monoclonal antibodies (mAbs) as a combinatory therapeutic. Nintedanib alone is not significantly cytotoxic to cells, but it causes cell cycle arrest and reverses EMT. By selecting for this sub-population of cells that are sensitive to Nintedanib, their subsequent eradication can then be more intricately directed. Since the anti-cancer application of EMT reversal agents are not fully developed, it is vital to investigate mechanisms that sensitize cancer cells to cytotoxic agents following EMT reversal....
Purpose: Metastases account for more than 90% of cancer deaths and hence, inhibiting metastasis i... more Purpose: Metastases account for more than 90% of cancer deaths and hence, inhibiting metastasis is an attractive therapeutic option. Accumulating evidences show that invasion and metastasis of ovarian carcinoma could be driven by Epithelial-Mesenchymal Transition (EMT), a developmental program being employed during carcinoma progression. As EMT is a reversible process, inhibiting or reversing EMT thus emerges as a potential anti-metastasis strategy. EMT is a spectrum consisting of the binary epithelial and mesenchymal states as well as transitional intermediate states. Therefore, reversing EMT from the mesenchymal state would result in the sliding along an EMT Spectrum. Currently, there is no quantitative tool available to assess the EMT spectrum. In this study, we describe a bioinformatics method for quantitating EMT. Experimental procedure: By using immunofluorescence staining of E-cadherin and N-cadherin, we defined the most epithelial and most mesenchymal cells in a panel of ova...
Epithelial ovarian cancer (EOC) is clinically heterogeneous, comprising different histological an... more Epithelial ovarian cancer (EOC) is clinically heterogeneous, comprising different histological and biological subtypes. Despite tumor heterogeneity, patient stratification is lacking and targeted therapeutic options for EOC are limited. Epithelial-mesenchymal transition (EMT), an important developmental process that allows the conversion of epithelial cells to mesenchymal phenotype, has been implicated in contributing to the molecular heterogeneity in EOC. Emerging evidence suggests that EMT in EOC may be associated with tumor progression and metastasis. We aim to decipher the underlying EMT mechanisms by using a panel of 42 EMT-phenotyped EOC cell lines. We found that Grainyhead-like 2 (GRHL2), a transcription factor, was expressed exclusively in EOC cells with epithelial phenotype by using qPCR, ELISA and western blots. EOC tumors with Mesenchymal molecular subtype also showed a decrease in GRHL2 expression. These data suggest that GRHL2 is required for maintaining cell polarity o...
The Asian Society of Gynecologic Oncology International Workshop 2014 on gynecologic oncology was... more The Asian Society of Gynecologic Oncology International Workshop 2014 on gynecologic oncology was held in Asan Medical Center, Seoul, Korea on the 23rd to 24th August 2014. A total of 179 participants from 17 countries participated in the workshop, and the up-to-date findings on the management of gynecologic cancers were presented and discussed. This meeting focused on the new trends in the management of cervical cancer, fertility-sparing management of gynecologic cancers, surgical management of gynecologic cancers, and recent advances in translational research on gynecologic cancers.
Frizzled family receptor 7 (FZD7), a Wnt signaling receptor, is associated with the maintenance o... more Frizzled family receptor 7 (FZD7), a Wnt signaling receptor, is associated with the maintenance of stem cell properties and cancer progression. FZD7 has emerged as a potential therapeutic target because it is capable of transducing both canonical and noncanonical Wnt signals. In this study, we investigated the regulatory pathway downstream of FZD7 and its functional roles. We found that FZD7 expression was crucial to the maintenance of the mesenchymal phenotype, anoikis resistance, and spheroid and tumor formation in ovarian cancer (OC). We identified TWIST1 as the crucial downstream effector of the FZD7 pathway. TWIST1, a basic helix loop helix transcription factor, is known to associate with mesenchymal and cancer stem cell phenotypes. Manipulating TWIST1 expression mimicked the functional consequences observed in the FZD7 model, and overexpression of TWIST1 partially rescued the functional phenotypes abolished by FZD7 knockdown. We further proved that FZD7 regulated TWIST1 expres...
AXL is a receptor tyrosine kinase that is often overexpressed in cancers. It contributes to patho... more AXL is a receptor tyrosine kinase that is often overexpressed in cancers. It contributes to pathophysiology in cancer progression and therapeutic resistance, making it an emerging therapeutic target. The first-in-class AXL inhibitor bemcentinib (R428/BGB324) was approved for treatment of acute myeloid leukaemia and was also reported to show selective sensitivity towards ovarian cancers (OC) with a Mesenchymal molecular subtype. In this study, we further explored AXLās role in mediating DNA damage responses by using OC as a disease model. AXL inhibition using R428 resulted in the increase of DNA damage with the concurrent upregulation of DNA damage response signalling molecules. Furthermore, AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress. Combinatory use of AXL and ATR inhibitors showed additive effects in OC. Through SILAC co-immunoprecipitation mass spectrometry, we identified a novel binding partner of AXL, SAM68, w...
Cancer cells exhibit phenotypic plasticity during epithelialāmesenchymal transition (EMT) and mes... more Cancer cells exhibit phenotypic plasticity during epithelialāmesenchymal transition (EMT) and mesenchymalāepithelial transition (MET) involving intermediate states. To study genome-wide epigenetic remodeling associated with EMT plasticity, we integrate the analyses of DNA methylation, ChIP-sequencing of five histone marks (H3K4me1, H3K4me3, H3K27Ac, H3K27me3 and H3K9me3) and transcriptome profiling performed on ovarian cancer cells with different epithelial/mesenchymal states and on a knockdown model of EMT suppressor Grainyhead-like 2 (GRHL2). We have identified differentially methylated CpG sites associated with EMT, found at promoters of epithelial genes and GRHL2 binding sites. GRHL2 knockdown results in CpG methylation gain and nucleosomal remodeling (reduction in permissive marks H3K4me3 and H3K27ac; elevated repressive mark H3K27me3), resembling the changes observed across progressive EMT states. Epigenetic-modifying agents such as 5-azacitidine, GSK126 and mocetinostat furth...
Epithelialāmesenchymal transition (EMT) encompasses dynamic changes in cellular organization from... more Epithelialāmesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by āthe EMT International Associationā (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data ge...
Cancer cells have many different behaviors from epithelial to mesenchymal forms. We report here t... more Cancer cells have many different behaviors from epithelial to mesenchymal forms. We report here that 36 distinct tumor cell lines regardless of EMT form or other features lack the ability to sense rigidity and will grow on soft surfaces. In the majority of lines, cells were missing at least one protein needed for rigidity sensing (primarily tropomyosin2.1 (Tpm2.1) but also PTPN12, FilaminA (FLNA), and myosinIIA) while all had high levels of Tpm3. In the few cases where the major rigidity sensing components were present, those tumor cells were not able to sense rigidity. Thus, we suggest that tumor cells can lose the ability to sense rigidity by many different means and that the loss of rigidity sensing is sufficient to cause the transformed phenotype that enables targeted treatments.
The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like... more The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial-mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial-mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis...
Background The plasticity along the epithelial-mesenchymal transition (EMT) spectrum has been sho... more Background The plasticity along the epithelial-mesenchymal transition (EMT) spectrum has been shown to be regulated by various epigenetic repertoires. Emerging evidence of local chromatin conformation changes suggests that regulation of EMT may occur at a higher order of three-dimensional genome level. Results We perform Hi-C analysis and combine ChIP-seq data across cancer cell lines representing different EMT states. We demonstrate that the epithelial and mesenchymal genes are regulated distinctively. We find that EMT genes are regulated within their topologically associated domains (TADs), with only a subset of mesenchymal genes being influenced by A/B compartment switches, indicating topological remodeling is required in the transcriptional regulation of these genes. At the TAD level, epithelial and mesenchymal genes are associated with different regulatory trajectories. The epithelial gene-residing TADs are enriched with H3K27me3 marks in the mesenchymal-like states. The mesenc...
Intrinsic intra-tumor heterogeneity (ITH) has been linked to worse patient outcomes. The developm... more Intrinsic intra-tumor heterogeneity (ITH) has been linked to worse patient outcomes. The development of spatial profiling technology has enabled the deciphering of ITH with multiple analysis readouts. Advanced ovarian clear cell carcinoma (OCCC), known to harbor ITH, is chemoresistant, poor prognostic, and possesses distinct molecular and histological characteristics. However, detailed spatial information of the nature of ITH within OCCC remains unclear. Here, we utilized the NanoString Digital spatial profiling (DSP) GeoMx platform to perform multiplex protein expression analysis on tumor samples of primary and colonic metastatic sites from one advanced OCCC patient. The spatial resolution revealed the existence of an epithelial-mesenchymal (EM) gradient within the metastatic tumor but not the primary tumor, and similar EM gradient was not observed within the primary tumor. The EM gradient exhibited a distinct pattern from the periphery to the core of the metastatic tumor. Compared...
This article describes a facile approach to sort millions of cells based on their migration speed... more This article describes a facile approach to sort millions of cells based on their migration speeds in 2D or 3D microenvironments. We validate the method using imaging, transcriptomics, and functional assays for cancer and immune cells. The tool is valuable to study new correlative and causative phenotypes associated with cell migration.
ABSTRACTWhereas phenotypic assays such as Boyden chambers and wound healing assays can easily be ... more ABSTRACTWhereas phenotypic assays such as Boyden chambers and wound healing assays can easily be employed to characterize the migratory potential of cells at the population level, few methods exist that can sort subpopulations of cells based on their migratory behaviour from an initial heterogeneous pool. In this paper, we present an approach to sort migratory cancer and immune cells based on their spontaneous migration in 2D and 3D microenvironments. Using this method, which is easy to implement and readily scalable, millions of live cells can be sorted based on their migratory characteristics and then subjected to downstream genomic, molecular and functional tests. We reveal that enrichment of the most migratory cytotoxic T lymphocytes yields a pool of cells with enhanced cytotoxicity against cancer cells. This new functional sorting method opens new avenues for the precise characterization of the mechanisms underlying hitherto unexplained heterogeneities in migratory phenotypes w...
Epithelial-mesenchymal transition (EMT) has long been associated with cancer progression and meta... more Epithelial-mesenchymal transition (EMT) has long been associated with cancer progression and metastasis. As a reversible process, it has the potential to be a target for cancer therapy. However, this concept of reversing EMT has not yet been widely explored in current treatment strategies. With the identification of Nintedanib as an EMT reversal agent, we aim to broaden the utility of this triple angiokinase inhibitor by identifying agents that show synergistic lethality with it, using monoclonal antibodies (mAbs) as a combinatory therapeutic. Nintedanib alone is not significantly cytotoxic to cells, but it causes cell cycle arrest and reverses EMT. By selecting for this sub-population of cells that are sensitive to Nintedanib, their subsequent eradication can then be more intricately directed. Since the anti-cancer application of EMT reversal agents are not fully developed, it is vital to investigate mechanisms that sensitize cancer cells to cytotoxic agents following EMT reversal....
Purpose: Metastases account for more than 90% of cancer deaths and hence, inhibiting metastasis i... more Purpose: Metastases account for more than 90% of cancer deaths and hence, inhibiting metastasis is an attractive therapeutic option. Accumulating evidences show that invasion and metastasis of ovarian carcinoma could be driven by Epithelial-Mesenchymal Transition (EMT), a developmental program being employed during carcinoma progression. As EMT is a reversible process, inhibiting or reversing EMT thus emerges as a potential anti-metastasis strategy. EMT is a spectrum consisting of the binary epithelial and mesenchymal states as well as transitional intermediate states. Therefore, reversing EMT from the mesenchymal state would result in the sliding along an EMT Spectrum. Currently, there is no quantitative tool available to assess the EMT spectrum. In this study, we describe a bioinformatics method for quantitating EMT. Experimental procedure: By using immunofluorescence staining of E-cadherin and N-cadherin, we defined the most epithelial and most mesenchymal cells in a panel of ova...
Epithelial ovarian cancer (EOC) is clinically heterogeneous, comprising different histological an... more Epithelial ovarian cancer (EOC) is clinically heterogeneous, comprising different histological and biological subtypes. Despite tumor heterogeneity, patient stratification is lacking and targeted therapeutic options for EOC are limited. Epithelial-mesenchymal transition (EMT), an important developmental process that allows the conversion of epithelial cells to mesenchymal phenotype, has been implicated in contributing to the molecular heterogeneity in EOC. Emerging evidence suggests that EMT in EOC may be associated with tumor progression and metastasis. We aim to decipher the underlying EMT mechanisms by using a panel of 42 EMT-phenotyped EOC cell lines. We found that Grainyhead-like 2 (GRHL2), a transcription factor, was expressed exclusively in EOC cells with epithelial phenotype by using qPCR, ELISA and western blots. EOC tumors with Mesenchymal molecular subtype also showed a decrease in GRHL2 expression. These data suggest that GRHL2 is required for maintaining cell polarity o...
The Asian Society of Gynecologic Oncology International Workshop 2014 on gynecologic oncology was... more The Asian Society of Gynecologic Oncology International Workshop 2014 on gynecologic oncology was held in Asan Medical Center, Seoul, Korea on the 23rd to 24th August 2014. A total of 179 participants from 17 countries participated in the workshop, and the up-to-date findings on the management of gynecologic cancers were presented and discussed. This meeting focused on the new trends in the management of cervical cancer, fertility-sparing management of gynecologic cancers, surgical management of gynecologic cancers, and recent advances in translational research on gynecologic cancers.
Frizzled family receptor 7 (FZD7), a Wnt signaling receptor, is associated with the maintenance o... more Frizzled family receptor 7 (FZD7), a Wnt signaling receptor, is associated with the maintenance of stem cell properties and cancer progression. FZD7 has emerged as a potential therapeutic target because it is capable of transducing both canonical and noncanonical Wnt signals. In this study, we investigated the regulatory pathway downstream of FZD7 and its functional roles. We found that FZD7 expression was crucial to the maintenance of the mesenchymal phenotype, anoikis resistance, and spheroid and tumor formation in ovarian cancer (OC). We identified TWIST1 as the crucial downstream effector of the FZD7 pathway. TWIST1, a basic helix loop helix transcription factor, is known to associate with mesenchymal and cancer stem cell phenotypes. Manipulating TWIST1 expression mimicked the functional consequences observed in the FZD7 model, and overexpression of TWIST1 partially rescued the functional phenotypes abolished by FZD7 knockdown. We further proved that FZD7 regulated TWIST1 expres...
AXL is a receptor tyrosine kinase that is often overexpressed in cancers. It contributes to patho... more AXL is a receptor tyrosine kinase that is often overexpressed in cancers. It contributes to pathophysiology in cancer progression and therapeutic resistance, making it an emerging therapeutic target. The first-in-class AXL inhibitor bemcentinib (R428/BGB324) was approved for treatment of acute myeloid leukaemia and was also reported to show selective sensitivity towards ovarian cancers (OC) with a Mesenchymal molecular subtype. In this study, we further explored AXLās role in mediating DNA damage responses by using OC as a disease model. AXL inhibition using R428 resulted in the increase of DNA damage with the concurrent upregulation of DNA damage response signalling molecules. Furthermore, AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress. Combinatory use of AXL and ATR inhibitors showed additive effects in OC. Through SILAC co-immunoprecipitation mass spectrometry, we identified a novel binding partner of AXL, SAM68, w...
Cancer cells exhibit phenotypic plasticity during epithelialāmesenchymal transition (EMT) and mes... more Cancer cells exhibit phenotypic plasticity during epithelialāmesenchymal transition (EMT) and mesenchymalāepithelial transition (MET) involving intermediate states. To study genome-wide epigenetic remodeling associated with EMT plasticity, we integrate the analyses of DNA methylation, ChIP-sequencing of five histone marks (H3K4me1, H3K4me3, H3K27Ac, H3K27me3 and H3K9me3) and transcriptome profiling performed on ovarian cancer cells with different epithelial/mesenchymal states and on a knockdown model of EMT suppressor Grainyhead-like 2 (GRHL2). We have identified differentially methylated CpG sites associated with EMT, found at promoters of epithelial genes and GRHL2 binding sites. GRHL2 knockdown results in CpG methylation gain and nucleosomal remodeling (reduction in permissive marks H3K4me3 and H3K27ac; elevated repressive mark H3K27me3), resembling the changes observed across progressive EMT states. Epigenetic-modifying agents such as 5-azacitidine, GSK126 and mocetinostat furth...
Epithelialāmesenchymal transition (EMT) encompasses dynamic changes in cellular organization from... more Epithelialāmesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by āthe EMT International Associationā (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data ge...
Cancer cells have many different behaviors from epithelial to mesenchymal forms. We report here t... more Cancer cells have many different behaviors from epithelial to mesenchymal forms. We report here that 36 distinct tumor cell lines regardless of EMT form or other features lack the ability to sense rigidity and will grow on soft surfaces. In the majority of lines, cells were missing at least one protein needed for rigidity sensing (primarily tropomyosin2.1 (Tpm2.1) but also PTPN12, FilaminA (FLNA), and myosinIIA) while all had high levels of Tpm3. In the few cases where the major rigidity sensing components were present, those tumor cells were not able to sense rigidity. Thus, we suggest that tumor cells can lose the ability to sense rigidity by many different means and that the loss of rigidity sensing is sufficient to cause the transformed phenotype that enables targeted treatments.
The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like... more The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial-mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial-mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis...
Background The plasticity along the epithelial-mesenchymal transition (EMT) spectrum has been sho... more Background The plasticity along the epithelial-mesenchymal transition (EMT) spectrum has been shown to be regulated by various epigenetic repertoires. Emerging evidence of local chromatin conformation changes suggests that regulation of EMT may occur at a higher order of three-dimensional genome level. Results We perform Hi-C analysis and combine ChIP-seq data across cancer cell lines representing different EMT states. We demonstrate that the epithelial and mesenchymal genes are regulated distinctively. We find that EMT genes are regulated within their topologically associated domains (TADs), with only a subset of mesenchymal genes being influenced by A/B compartment switches, indicating topological remodeling is required in the transcriptional regulation of these genes. At the TAD level, epithelial and mesenchymal genes are associated with different regulatory trajectories. The epithelial gene-residing TADs are enriched with H3K27me3 marks in the mesenchymal-like states. The mesenc...
Intrinsic intra-tumor heterogeneity (ITH) has been linked to worse patient outcomes. The developm... more Intrinsic intra-tumor heterogeneity (ITH) has been linked to worse patient outcomes. The development of spatial profiling technology has enabled the deciphering of ITH with multiple analysis readouts. Advanced ovarian clear cell carcinoma (OCCC), known to harbor ITH, is chemoresistant, poor prognostic, and possesses distinct molecular and histological characteristics. However, detailed spatial information of the nature of ITH within OCCC remains unclear. Here, we utilized the NanoString Digital spatial profiling (DSP) GeoMx platform to perform multiplex protein expression analysis on tumor samples of primary and colonic metastatic sites from one advanced OCCC patient. The spatial resolution revealed the existence of an epithelial-mesenchymal (EM) gradient within the metastatic tumor but not the primary tumor, and similar EM gradient was not observed within the primary tumor. The EM gradient exhibited a distinct pattern from the periphery to the core of the metastatic tumor. Compared...
Uploads
Papers by Ruby Huang