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Chan, Leo, Haohai Zhang, William Rice, et al. "Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery." Journal of Immunology 200, no. 1_Supplement (2018): 120.1. http://dx.doi.org/10.4049/jimmunol.200.supp.120.1.
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Abstract Hybridoma screening is a highly important process that can identify potential clones from hybridoma cultures against the desired target antigen. Traditional screening methods using ELISA and flow cytometry presented technical issues such as limited accuracy, reduced high-throughput capability, increased time and cost. Here, we demonstrate a novel cell-based high-throughput screening method using the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on CHO cells. Both target CHO and CFSE-stained wild type were co-cultured to simultaneously detect target antibodies and nonspecific binding, which can eliminate the need to produce a separate control sample for each hybridoma supernatant. Next, the image cytometer was used to screen 672 hybridoma supernatants to develop the screening workflow. The Celigo was used to quickly image and analyze antibody binding using Hoechst, Alexa Fluor 594 and CFSE fluorescence to identify high, medium, and low binding hits. In addition, the Celigo was used to measure the binding affinities of the target antibodies to membrane-bound CD39. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5 ng/mL. We propose that this method will greatly improve screening technologies and facilitate more efficient antibody discovery.
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Pierce, Mackenzie, Yongyang Huang, and Bo Lin. "Abstract 485: Using the Celigo™ Image Cytometer for effectively screening rAAV gene delivery methods." Cancer Research 85, no. 8_Supplement_1 (2025): 485. https://doi.org/10.1158/1538-7445.am2025-485.
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Abstract To develop effective rAAV gene delivery vectors, scientists need to consider multiple elements, such as the promoter, insertion location, genetic target, and target gene to name a few. Green fluorescent protein (GFP), or other detection markers are often used to measure vector transduction and gene expression efficiency. Transduced GFP samples can be analyzed using fluorescent microscopy and flow cytometry, however, both methods can be quite time consuming, labor intensive, and lack high-throughput capabilities to kinetically monitor gene expression. In this work, we developed and employed the Celigo™ Image Cytometer to screen different gene delivery vectors by comparing GFP expression level in SupT1 cells at 24-, 48-, 72-, and 96-hours following transduction. Using the Celigo™ we were able to monitor transient and integrated GFP expression amongst multiple gene delivery vectors. For transient expression monitoring, SB-P-AA-101-07 displayed stronger expression levels at 24- and 48-hours. For integrated expression monitoring, both plasmid and rAAV with integration element exhibited over ∼90%+ GFP transduction efficiency by 96-hours. In summary, the Celigo™ may prove useful for researchers developing and screening gene delivery vectors in a high throughput manner. Note: SB-P-AA-101-07 (from Revvity Health Sciences, Inc.) is for research use only. Not for use in diagnostic procedures. Citation Format: Mackenzie Pierce, Yongyang Huang, Bo Lin. Using the Celigo™ Image Cytometer for effectively screening rAAV gene delivery methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 485.
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Lin, Mong-Shang, Carolina Franco Nitta, Mackenzie Pierce, Bo Lin, Leo L. Chan, and Jessie Ni. "Abstract 3168: High-throughput hybridoma monoclonality characterization using the Celigo™ Image Cytometer." Cancer Research 85, no. 8_Supplement_1 (2025): 3168. https://doi.org/10.1158/1538-7445.am2025-3168.
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Abstract Hybridomas are hybrid cells produced by fusing an antibody producing B cell with a myeloma cell to produce a specific monoclonal antibody. In the process of generating stable clones, developers are often required to perform several rounds of subcloning by plating cultures with a density of a single cell per well. The wells with a single colony are then selected to produce the target antibodies. Traditionally, this lengthy process was heavily relied on researchers with extensive training and experience; however, even experienced researchers may suffer from human error of selecting clones that are not truly monoclonal, thus potentially delaying antibody development and increasing costs. In this work, we demonstrate a high-throughput solution that has successfully established monoclonality of our hybridoma by employing the Celigo™ Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA) with the robotic arm accessory to screen potential candidates for downstream antibody production under a GMP environment. The Celigo™ is a plate-based, whole well, high-throughput image cytometry system with brightfield and fluorescence imaging capabilities. In combination with the robotic arm, automation can be implemented to further streamline the monoclonality workflow, and subsequently reduce subcloning personnel time by 25-50%, while also minimizing human handling errors by at least 10-fold. Following imaging acquisition, an in-house monoclonality analytical software was utilized to provide concrete documentation for every clone selected for production. Lastly, we demonstrated that time for hybridoma development in clone confirmation screening as well as subcloning could be further improved by incorporating ClonePix technology. Citation Format: Mong-Shang Lin, Carolina Franco Nitta, Mackenzie Pierce, Bo Lin, Leo L. Chan, Jessie Ni. High-throughput hybridoma monoclonality characterization using the Celigo™ Image Cytometer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3168.
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Liao, Wen-Chieh, Julia Abdalla, Carolina Franco Nitta, Mackenzie Pierce, Bo Lin, and Jessie Ni. "Abstract 5473: Development of a novel fluorescent ELISpot assay using the CeligoTMImage Cytometer." Cancer Research 85, no. 8_Supplement_1 (2025): 5473. https://doi.org/10.1158/1538-7445.am2025-5473.
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ELISpot and its variant, fluorescent ELISpot (FluoroSpot), are commonly used methodologies that monitor and detect cytokine and/or antibody secretion from peripheral blood mononuclear cells (PBMCs). Conventional ELISpot and FluoroSpot assays utilize PVDF-membrane plates, which require labor-intensive washing steps that may introduce variability in performance and results. Here, we have developed a novel FluoroSpot assay using polystyrene-, clear-bottom microplates with simplified automated washing steps to minimize operator variability and error. In this work, we assessed multiple critical cellular immune-related activities, including IL-6, IFN-g, and TNF-a secretion in PBMCs using this novel FluoroSpot methodology in comparison to the conventional ELISpot assay. To detect cytokine secretion of IL-6, IFN-g, and TNF-a, the Celigo™ Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA) was incorporated to capture whole-well images of fluorescently labeled detection antibodies. Results for both methodologies were comparable to one another, validating the use of polystyrene plates on Celigo™ as opposed to PVDF-membrane plates. In addition to FluoroSpot detection, the Celigo™ was used as a quality control check to provide a comprehensive analysis of live cell distribution prior to performing the FluoroSpot assay. Live cell imaging results were used to correlate downstream cytokine-releasing cell spot formation results. Combinational use of the novel FluoroSpot polystyrene plate with the Celigo™ reduced operator variability and errors, while streamlining a multiparametric analysis to identify the number of fluorescent spots, spot morphology (size), and whole-well mean fluorescence intensity values. This methodology may prove useful for researchers interested in monitoring and detecting cytokine and/or antibody-releasing PBMCs. Citation Format: Wen-Chieh Liao, Julia Abdalla, Carolina Franco Nitta, Mackenzie Pierce, Bo Lin, Jessie Ni. Development of a novel fluorescent ELISpot assay using the CeligoTMImage Cytometer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5473.
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Cribbes, Scott, Sarah Kessel, Scott McMenemy, Jean Qiu, and Leo Li-Ying Chan. "A Novel Multiparametric Drug-Scoring Method for High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 5 (2017): 547–57. http://dx.doi.org/10.1177/2472555217689884.
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Three-dimensional (3D) tumor models have been increasingly used to investigate and characterize cancer drug compounds. The ability to perform high-throughput screening of 3D multicellular tumor spheroids (MCTS) can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Previously, the Celigo Image Cytometer has demonstrated a novel method for high-throughput screening of 3D multicellular tumor spheroids. In this work, we employed the Celigo Image Cytometer to examine the effects of 14 cancer drug compounds on 3D MCTS of the glioblastoma cell line U87MG in 384-well plates. Using parameters such as MCTS diameter and invasion area, growth and invasion were monitored for 9 and 3 d, respectively. Furthermore, fluorescent staining with calcein AM, propidium iodide, Hoechst 33342, and caspase 3/7 was performed at day 9 posttreatment to measure viability and apoptosis. Using the kinetic and endpoint data generated, we created a novel multiparametric drug-scoring system for 3D MCTS that can be used to identify and classify potential drug candidates earlier in the drug discovery process. Furthermore, the combination of quantitative and qualitative image data can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects. The 3D MCTS-based multiparametric scoring method described here can provide an alternative screening method to better qualify tested drug compounds.
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Zhang, Haohai, Leo Li-Ying Chan, William Rice, et al. "Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer." Journal of Immunological Methods 447 (August 2017): 23–30. http://dx.doi.org/10.1016/j.jim.2017.04.003.
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Chan, Leo, Kai W. Wucherpfennig, and Lucas De Andrade. "Long term time-course monitoring of NK cell-mediated ADCC using the Celigo Image Cytometer." Journal of Immunology 204, no. 1_Supplement (2020): 88.4. http://dx.doi.org/10.4049/jimmunol.204.supp.88.4.
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Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) assay has been widely performed for immuno-oncological research. Traditionally, ADCC assays are conducted by measuring the amount of released Chromium, calcein AM, or Lactate dehydrogenase (LDH) molecules after the target cancer cells are killed by effector/antibody pair. These methods can be inaccurate due to indirect measurement of supernatant at the end point of 4 hour, however, there is a need to characterize the effects of antibody-dependent cytotoxicity for longer than 72 hours. In this work, we demonstrated the time-course monitoring of NK cell-mediated ADCC of A375 cells in the presence or absence of IL2 and antibodies for 76 hours. First, A375-ZsGreen cells were seeded into a 96-well plate with and without target antibodies. Next, NK cells with and without IL2 were added to the wells at 10:1, 5:1, 2:1, and 1:1 E:T ratios. The co-culture was allowed to incubate for 76 hours and the image cytometer was used to scan and analyze at times from 0 to 76 hours. The software was able to count individual cells as well as confluence % at each time point by segmenting highly fluorescent objects in the images. We were able to show time-dependent ADCC killing with decrease in ZsGreen positive cells as the read-out. In addition, the uniformity of cell killing can be observed in the Celigo obtained whole well images. One of the most important findings was that there was regrowth of target cells after the initial 30 hours of cell killing, indicating the activated NK cells without antibodies did not eradicate the cancer cells. Therefore, the ability to perform long term time-course monitoring of ADCC is highly important to better characterize the effects of target antibodies on cell killing function.
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Chan, Leo, Kelsey McCulley, and Pinaki Banerjee. "A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity." Journal of Immunology 196, no. 1_Supplement (2016): 143.11. http://dx.doi.org/10.4049/jimmunol.196.supp.143.11.
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Abstract Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First the live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a flat- bottom 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was implied to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined using the “self-referencing” calculation, which could be a more direct method for assessing cytotoxicity than 51Cr release. The donor PBMCs were added to each well with different Calcein-stained target cells at Effector-to-Target (E:T) ratios. The 96-well microplate was scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. The proposed image cytometry method can scan and analyze in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.
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Jiang, Wen, W. Nathaniel Brennen, Samuel R. Denmeade, and Daniel L. Thorek. "Abstract 6055: Cell confluence growth tracking as a supplement assay for clonogenic assay in prostate cancer radiotherapy combination evaluation." Cancer Research 82, no. 12_Supplement (2022): 6055. http://dx.doi.org/10.1158/1538-7445.am2022-6055.
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Abstract Background: Radiation therapy is a pillar of cancer care, and over 50% of all cancer patients will receive a form of this treatment. There is great interest in the capacity to improve radiation response and to de-escalate dose through combination pharmacological strategies. Evaluating these methods is a persistent challenge to the field. The clonogenic assay has been the gold standard assay in radiobiology research to evaluate the ability of a single cell to grow into a colony after different radiation treatments. However, the assay is laborious, time consuming and sensitive to environmental conditions. Also, the low cell seeding density may pose challenges for cells to proliferate, and extra conditioned culture medium is required for certain cell lines. Here, we have used the Celigo imaging cytometer to track cell confluence growth day by day, to demonstrate its comparable ability with the clonogenic assay to reveal differences in cell proliferation ability after different radiation treatments. Methods: Firstly, confluence measurement consistency was validated by repeatedly measuring the same wells for 6 times (Celigo 200 BFFL). Next, cell confluence growth tracking was compared with the clonogenic assay using 4 prostate cancer cell lines (LNCaP, PC3, 22Rv1, and LN95), after X-ray treatment and X-ray in combination with androgen receptor (AR)-inhibiting enzalutamide treatment. Treated cells were collected and seeded to 12-well plates followed by daily checks with Celigo. The Celigo imaging cytometer can image each well and conduct automatic image segmentation to calculate cell confluence reported as a percentage of well area. Daily cell confluence data was used to draw cell growth curves. Results: The consistency of cell confluence measurements was validated with a coefficient of variation less than 5%. In comparing cell confluence growth with clonogenic assay, with the increase of X-ray dose (0Gy, 2Gy, and 4Gy), the degenerating cell proliferation ability was consistently observed in all 4 cell lines with both assays. Additionally, X-ray in combination with enzalutamide treated LNCaP cells demonstrated decreased cell proliferation ability than X-ray alone treated cells, as demonstrated by both cell confluence tracking and clonogenic assay. However, enzalutamide did not affect the proliferation ability of X-ray treated AR-negative PC3 cells, and was confirmed in both assays. The radiopotentiation effect of enzalutamide was also observed in AR-expressing 22Rv1 and LN95 cells with clonogenic assay, but not in 22Rv1 cells with cell confluence tracking. Conclusion: Cell confluence tracking with Celigo allows daily in situ monitoring of cells and a higher cell seeding density than the clonogenic assay. It is able to provide additional evidence of cell proliferation to supplement the traditional clonogenic assay in radiobiology research. Citation Format: Wen Jiang, W. Nathaniel Brennen, Samuel R. Denmeade, Daniel L. Thorek. Cell confluence growth tracking as a supplement assay for clonogenic assay in prostate cancer radiotherapy combination evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6055.
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Kessel, Sarah, Scott Cribbes, Olivier Déry, et al. "High-Throughput 3D Tumor Spheroid Screening Method for Cancer Drug Discovery Using Celigo Image Cytometry." SLAS TECHNOLOGY: Translating Life Sciences Innovation 22, no. 4 (2016): 454–65. http://dx.doi.org/10.1177/2211068216652846.
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Oncologists have investigated the effect of protein or chemical-based compounds on cancer cells to identify potential drug candidates. Traditionally, the growth inhibitory and cytotoxic effects of the drugs are first measured in 2D in vitro models, and then further tested in 3D xenograft in vivo models. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed under a 3D environment. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids using the Celigo image cytometer. First, optimal seeding density for tumor spheroid formation was determined by investigating the cell seeding density of U87MG, a human glioblastoma cell line. Next, the dose–response effects of 17-AAG with respect to spheroid size and viability were measured to determine the IC50 value. Finally, the developed high-throughput method was used to measure the dose response of four drugs (17-AAG, paclitaxel, TMZ, and doxorubicin) with respect to the spheroid size and viability. Each experiment was performed simultaneously in the 2D model for comparison. This detection method allowed for a more efficient process to identify highly qualified drug candidates, which may reduce the overall time required to bring a drug to clinical trial.
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Kessel, Sarah, Scott Cribbes, Surekha Bonasu, Jean Qiu, and Leo Li-Ying Chan. "Real-Time Apoptosis and Viability High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 2 (2017): 202–10. http://dx.doi.org/10.1177/2472555217731076.
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Three-dimensional tumor spheroid models have been increasingly used to investigate and characterize cancer drug compounds. Previously, the Celigo image cytometer has demonstrated its utility in a high-throughput screening manner for evaluating potential drug candidates in a 3D multicellular tumor spheroid (MCTS) primary screen. In addition, we have developed real-time kinetic caspase 3/7 apoptosis and propidium iodide viability 3D MCTS assays, both of which can be used in a secondary screen to better characterize the hit compounds. In this work, we monitored the kinetic apoptotic and cytotoxic effects of 14 compounds in 3D MCTS produced from the glioblastoma cell line U87MG in 384-well plates for 9 days. The kinetic results allowed the categorization of the effects from 14 drug compounds into early and late cytotoxic, apoptotic, cytostatic, and no effects. The real-time apoptosis and viability screening method can serve as an improved secondary screen to better understand the mechanism of action of these potential drug candidates identified from the primary screen, allowing one to identify a more qualified drug candidate and streamline the drug discovery process of research and development.
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Kessel, Sarah, Scott Cribbes, Surekha Bonasu, William Rice, Jean Qiu, and Leo Li-Ying Chan. "Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer." Cytometry Part A 91, no. 9 (2017): 883–92. http://dx.doi.org/10.1002/cyto.a.23143.
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Chan, Leo. "Novel plate-based detection method for T cell activation/proliferation, migration, and cytotoxicity assay using image cytometry." Journal of Immunology 202, no. 1_Supplement (2019): 130.8. http://dx.doi.org/10.4049/jimmunol.202.supp.130.8.
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Abstract The field of cancer immunotherapy has grew significantly due to numerous successful trials in cancer vaccines, antibody-based treatments, CAR T cell therapy, and other immunotherapies. Specifically for T cell-based assays, they have been traditionally analyzed using ELISA or ELISpot to detect specific cytokines concentration for measuring T cell responses. Other methods such as T cell activation/proliferation, migration, and cytotoxicity assays have employed flow cytometry or microscopy to understand the functionality of T cells. In the recent years, image cytometry has been used to develop robust assays to monitor T cell immune response, which is highly critical for immuno-oncology research. In this work, we demonstrate the use of Celigo Image Cytometer to perform high-throughput in vitro T cell-based functional assays. Three direct cell counting methods are developed for label-free T cell activation and proliferation measurement, T cell migration assay, as well as T cell-mediated cytotoxicity. First, primary T cells are seeded into a 96-well plate and stimulated with antibodies or co-culture with cancer cells. The cells are directly counted in the well over 48 hours in bright field images. Next, T cells are seeded in a transwell with chemokines in the bottom wells. As the T cells migrate through and drop to the bottom, the cells are directly counted using bright field or fluorescence with Hoechst staining. Finally, T cell-mediated cytotoxicity assay is performed by co-culturing CD8 T cells with calcein- or fluorescent protein-labeled cancer cells. As target cancer cells are killed, the fluorescence dissipates, and by counting the changes in the number of “live” target cells, the cytotoxicity percentages are measured.
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Chan, Leo, Yu-Jun Sun, Yi-Chun Chen, Wei-Kai Hua, and Sariena Wu. "Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method." Journal of Immunology 210, no. 1_Supplement (2023): 250.01. http://dx.doi.org/10.4049/jimmunol.210.supp.250.01.
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Abstract In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Traditionally, in vitro CAR-T cell-mediated cytotoxicity is assessed using release assays such as 51Cr (radioactivity), calcein (fluorescence), and LDH (enzymatic). Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo high-throughput plate-based Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. Performing time- and E:T ratio-dependent CAR-T cell-mediated cytotoxicity assays, the GFP-based method demonstrated higher sensitivity in detecting the level of cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions. None
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McDonald, James, Leo Li-Ying Chan, Jocelyn Reader, Sergio Mojica, and Mackenzie Pierce. "Abstract 5930: Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method." Cancer Research 84, no. 6_Supplement (2024): 5930. http://dx.doi.org/10.1158/1538-7445.am2024-5930.
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Abstract Ovarian cancer accounts for approximately 6% of all cancer death in women and has one of the highest mortality rates out of all gynecologic malignancies in the United States. Ovarian cancer can exhibit innate or acquired chemoresistance behavior, thus it is critical to identify novel therapeutic drug candidates. One of important characteristics of identifying potential chemotherapy drug candidate is the ability to induce apoptosis or programmed cell death in the target cancer cells. In order to measure apoptosis, various biomarkers are commonly employed such as Caspase 3, 7, 8 or 9, as well as Annexin V, which are all part of the cascade in apoptotic intrinsic pathway. However, typical fluorescent staining for image cytometry is not multiplexed that required multiple steps in order to determine the different type of apoptotic populations. In this work, we demonstrate a multiplexing apoptosis detection method to investigate the apoptotic effects of EP4A1, EP4A2, Paclitaxel, and Carboplatin on Kuramochi ovarian cancer cells. We employed the use of a high-throughput plate-based image cytometer (Celigo, Revvity Health Sciences Inc.) to image and analyze drug-treated Kuramochi cells stained with Annexin V-APC, Caspase 3/7 (488), and propidium iodide (PI) to assess the early- and late-stage apoptosis, as well as necrosis. We expected to see an increase in dual staining with the addition of EP4A to paclitaxel and carboplatin; however, the more interesting data was observed by separating single positive cell populations (Caspase, Annexin or PI only) from dual positive cell populations (Annexin + PI, Caspase + PI, Annexin +PI). The results show that treatment with EP4A lead to a time dependent increase in Caspase 3 cleavage but only in the single positive cell population. This trend was also observed in Annexin V single positive cell populations which is an indication of early apoptosis. The additive effect observed with the addition of EP4A was not seen in the dual positive cell populations. The high throughput nature of the image cytometry also allowed us to easily and quickly determine the optimal treatment conditions for this experiment. We were able to record multiple time points and not only capture proliferation, but also simultaneously collect data on caspase cleavage, Annexin-V staining and PI staining on multiple concentrations of compounds in monotherapy, dual therapy, and triple therapy combinations. Typically, it may be difficult to test multiple conditions in duplicate and triplicate using flow cytometry, while Celigo Image Cytometry may be used to obtain more robust data, and simultaneously acquire data of proliferation and multiplex staining with Annexin-V, PI and Caspase 3/7. Citation Format: James McDonald, Leo Li-Ying Chan, Jocelyn Reader, Sergio Mojica, Mackenzie Pierce. Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5930.
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Kessel, Sarah L., and Leo Li-Ying Chan. "A High-Throughput Image Cytometry Method for the Formation, Morphometric, and Viability Analysis of Drug-Treated Mammospheres." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 7 (2020): 723–33. http://dx.doi.org/10.1177/2472555220922817.
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The nonadherent mammosphere assay has been commonly used to investigate cancer stem cell activities in breast cancers that have the ability to form tumorspheres and maintain tumor growth. The sphere formation step is critical, in that it enables the construction of the mammosphere models for downstream assays. The mammosphere assay has also been used to assess the effects of drug treatment on the tumorspheres formed from primary cancer cells or cell lines. Traditionally, the mammosphere formation has been evaluated by standard microscopy systems that required external software for additional analyses. However, this method can be time-consuming and low-throughput, thus impractical for high-throughput characterization of mammosphere models and screening for potential therapeutic cancer drugs. To overcome these challenges, we developed a plate-based high-throughput method to rapidly analyze mammospheres in whole wells using the Celigo Image Cytometer. The method is employed to characterize mammosphere formation and morphology for adherent and nonadherent propagation of four breast cancer cell lines (MCF7, MDA-MB-436, MDA-MB-231, and SKBR3). Next, the dose-dependent effects of four small molecule drugs (doxorubicin, paclitaxel, 8-quinolinol, and salinomycin) are characterized based on sphere formation and viability stained with calcein AM and propidium iodide. We observed growth and morphometric differences between adherent and nonadherent propagation of the four cell lines. Furthermore, drug treatments induced various effects on mammosphere formation, morphology, and viability. The proposed image cytometry method provides a useful tool suitable for high-throughput characterization and analysis of mammospheres, which can improve assay efficiency when investigating the formation capabilities and drug-induced cytotoxicity effects.
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Bell, Jordan, Shilpaa Mukundan, Matthew Teryek, Bo Lin, Biju Parekkadan, and Leo Chan. "Abstract 184: High-throughput chemotherapeutic drug screening of tumor spheroids with individual spheroid results using image cytometry." Cancer Research 82, no. 12_Supplement (2022): 184. http://dx.doi.org/10.1158/1538-7445.am2022-184.
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Abstract Three-dimensional cancer models have gained popularity for in vitro studies of chemotherapeutic compounds by providing a more physiologically relevant analog of gas, nutrient, and drug diffusion throughout the tumor microenvironment. Some 3D assays are performed to study individual spheroids over time, where a majority of these assays rely on maintaining a single spheroid in each well of a 96-well round-bottom ultra-low attachment plate, limiting the number of spheroids in a study. Other assays may gather population-level data from large ensembles of spheroids grown together, but the information about individual differences amongst the spheroids is lost. Important kinetic information may also be lost for destructive endpoint assays such as MTS or MTT. Here, we describe the development of a 3D image cytometry assay that is capable of generating kinetic data for thousands of breast cancer spheroids at the individual level. T47D spheroids are grown and maintained in a 24-well Aggrewell࣪400 plate and imaged using the Celigo image cytometer. Each well contains more than 1000 subwells that both aid in spheroid formation and constrain each spheroid to a specific location. Using the spheroid location data, we are able to track and monitor the growth of each spheroid over 7 days. Furthermore, we investigate the dose-dependent effects on spheroid viability of 6 anti-cancer drugs (Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen) using calcein AM and propidium iodide (PI). To validate the viability measurement results, we utilize the CellTiter96® MTS assay as an orthogonal method to compare the dose-dependent trends using both the calcein AM and PI fluorescence intensities as well as the spheroid sizes. This work may lay a foundation for the investigation of other spheroids, organoids, or tissue samples, significantly increasing the number of spheroids analyzed per condition, improving the statistical analysis, and adding more parameters to further analyze the spheroids. These improvements may be especially helpful for spheroids grown from patient-derived or otherwise heterogeneous cell populations Citation Format: Jordan Bell, Shilpaa Mukundan, Matthew Teryek, Bo Lin, Biju Parekkadan, Leo Chan. High-throughput chemotherapeutic drug screening of tumor spheroids with individual spheroid results using image cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 184.
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Chan, Leo Li-Ying, Yu-Jun Sun, Yi-Chun Chen, Wei-Kai Hua, and Sariena Chiung-Yuan Wu. "Abstract 5326: Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method." Cancer Research 83, no. 7_Supplement (2023): 5326. http://dx.doi.org/10.1158/1538-7445.am2023-5326.
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Abstract In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Traditionally, in vitro CAR-T cell-mediated cytotoxicity is assessed using release assays such as 51Cr (radioactivity), calcein (fluorescence), and LDH (enzymatic). However, release assays indirectly measure cell death via molecules released in supernatant and typically limited to only endpoint assays. In addition, handling and disposing of 51Cr hazardous materials is less preferred. Luciferase reporter assay, although highly sensitive, has similar drawbacks as the release assays. Finally, flow cytometry method can directly measure cell death and viability, but can be time-consuming and requires a large number of CAR-T cells when the effector-to-target (E:T) ratio is high. Furthermore, additional steps are required for adherent cells that require trypsinization. Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo high-throughput plate-based Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. Performing time- and E:T ratio-dependent CAR-T cell-mediated cytotoxicity assays, the GFP-based method demonstrated higher sensitivity in detecting the level of cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions. Citation Format: Leo Li-Ying Chan, Yu-Jun Sun, Yi-Chun Chen, Wei-Kai Hua, Sariena Chiung-Yuan Wu. Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5326.
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Kim, Olga, Madison Butler, Ying Pang, et al. "EXTH-03. COMBINED TOP1 AND PARP INHIBITION ENFORCES GENOMIC INSTABILITY AND CELL DEATH IN PTEN-DEFICIENT GLIOBLASTOMA." Neuro-Oncology 23, Supplement_6 (2021): vi163—vi164. http://dx.doi.org/10.1093/neuonc/noab196.642.
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Abstract BACKGROUND Glioblastoma is an aggressive brain tumor with high mortality. The development of new therapies is critical for improving patient outcomes. LMP400, a novel topoisomerase I (TOP1) inhibitor, traps TOP1 cleavage complexes, thereby generating DNA damage. Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair responses triggered by TOP1 inhibition. Niraparib is a potent PARP inhibitor that can cross the blood-brain barrier. Loss of phosphatase and tensin homolog (PTEN) occurs in 40% of GBM patients and is known to promote DNA damage repair deficiency. Here, we hypothesize that PTEN loss presents a vulnerability to a combined induction of DNA damage and inhibition of repair mechanisms. METHODS Human glioblastoma cells (U251, SNB-75, SF-295, LN18) and patient-derived glioblastoma stem cells (GSC923 and GSC827) were treated with LMP400 and/or Niraparib. Cell viability and apoptosis were examined using Celigo image cytometer and Annexin V/PI assay at 72h after treatment. Single clones after PTEN knockdown using shRNA were isolated after puromycin selection. For planned studies of PTEN knockout, sgRNA plasmids targeting PTEN will be transiently transfected and GFP-positive single KO clones will be isolated. PTEN will be restored in PTEN-null cells using lentiviral transduction. RESULTS CRISPR-Cas9 KO screening in GSC923 cells suggests that LMP400 is unlikely a substrate for ABC transporters. LMP400 and Niraparib synergistically induced cytotoxic effects in U251, SF-295, GSC923, GSC827 cells lacking PTEN expression. Combined LMP400/Niraparib led to increased expression of gamma-H2AX, cleaved caspase 3 and PARP, indicative of enhanced DNA damage and cell death. CONCLUSION LMP400 and Niraparib act synergistically to target PTEN-deficient glioblastoma by inducing DNA damage and cell death. These results will be further verified in isogenic cells in vitro as well as in vivo in a mouse model driven by PTEN deletion which would strongly support a novel therapeutic strategy in a subset of glioblastoma with PTEN loss.
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Mukherjee, Joydeep, Carolina Franco Nitta, Mackenzie Pierce, et al. "Abstract 3103: Alternative cell counting and viability detection using NexGreen/PI fluorescent stain on multiple low- and high-throughput image cytometry platforms." Cancer Research 84, no. 6_Supplement (2024): 3103. http://dx.doi.org/10.1158/1538-7445.am2024-3103.
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Abstract In the past decade, the increase in cell and gene therapy products such as biologics, antibodies, and CAR-T has significantly increased the need for precise, robust, and consistent cell counting and viability measurements. Simplified and automated workflows benefit from assays that measure sample characteristics independent of their buffer, media, or storage conditions. Reliable and robust results are thus required to maintain quality control of downstream processes. In this work, we demonstrate a novel fluorescence-based dye, NexGreen/PI, capable of live and dead cell detection comparable to AO/PI in multiple cell buffer/media conditions. Jurkat cells of different viabilities (low, intermediate, and high) were stained in parallel with either NexGreen/PI or AO/PI for 1-2 minutes and measured on multiple image cytometer platforms including low-throughput (Cellometer K2, Auto2000, and Spectrum) and high-throughput (Cellaca MX, Cellaca PLX, and Celigo) instruments. Both NexGreen/PI and AO/PI dyes are used as equal volumes added to cell samples (ex: 20 uL cells + 20 uL dye). Additionally, we tested if difficult to analyze cells in “spent” media (typically with a lower pH) or in PBS can be stained using NexGreen/PI compared to AO/PI. Results on the high-throughput platforms show that NexGreen/PI staining can discriminate between live and dead Jurkats, analogous to AO/PI in samples of low (10-25%), intermediate (40-60%), and high (80-95%) viabilities. Measured viability of all samples using NexGreen/PI was within 5% of the paired AO/PI-stained samples. Cell concentration results with either NexGreen/PI or AO/PI also show counts with less than 6% difference. Low-throughput instrument results are similar and show that viabilities of Jurkat cells match within 5% of each other independent of sample (low, intermediate, or high viability), while cell concentrations differ up to 6% when comparing NexGreen/PI and AO/PI. Cells in “spent” media or washed and resuspended in PBS can exhibit low AO signal intensity that can be difficult to detect in a robust fashion. NexGreen/PI-stained cells, however, showed an unaltered fluorescence signal in these “tricky” cell samples. In this work, we demonstrated the capability of NexGreen/PI to measure cell counts and viability using low- and high-throughput instruments, which enables fit-for-purpose for a wide range of samples. Thus, our experiments demonstrate that NexGreen/PI is scalable and can be of significant value for use in automation and high-throughput systems where multiple samples in different mediums can be precisely measured. Citation Format: Joydeep Mukherjee, Carolina Franco Nitta, Mackenzie Pierce, Sopaul Hem, Daniel Browe, Dmitry Kuksin, Leo Li-Ying Chan. Alternative cell counting and viability detection using NexGreen/PI fluorescent stain on multiple low- and high-throughput image cytometry platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3103.
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Chan, Leo Li-Ying, Tim Smith, Kendra A. Kumph, et al. "A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry." Cytotechnology 68, no. 5 (2016): 2015–25. http://dx.doi.org/10.1007/s10616-016-0015-x.
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St Clair, Laura A., Leo Li-Ying Chan, Adam Boretsky, Bo Lin, Michael Spedding, and Rushika Perera. "High-Throughput SARS-CoV-2 Antiviral Testing Method Using the Celigo Image Cytometer." Journal of Fluorescence, June 13, 2023. http://dx.doi.org/10.1007/s10895-023-03289-x.
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AbstractThe COVID-19 pandemic has created a worldwide public health crisis that has since resulted in 6.8 million reported deaths. The pandemic prompted the immediate response of researchers around the world to engage in rapid vaccine development, surveillance programs, and antiviral testing, which resulted in the delivery of multiple vaccines and repurposed antiviral drug candidates. However, the emergence of new highly transmissible SARS-CoV-2 variants has renewed the desire for discovering new antiviral drug candidates with high efficacy against the emerging variants of concern. Traditional antiviral testing methods employ the plaque-reduction neutralization tests (PRNTs), plaque assays, or RT-PCR analysis, but each assay can be tedious and time-consuming, requiring 2–3 days to complete the initial antiviral assay in biologically relevant cells, and then 3–4 days to visualize and count plaques in Vero cells, or to complete cell extractions and PCR analysis. In recent years, plate-based image cytometers have demonstrated high-throughput vaccine screening methods, which can be adopted for screening potential antiviral drug candidates. In this work, we developed a high-throughput antiviral testing method employing the Celigo Image Cytometer to investigate the efficacy of antiviral drug candidates on SARS-CoV-2 infectivity using a fluorescent reporter virus and their safety by measuring the cytotoxicity effects on the healthy host cell line using fluorescent viability stains. Compared to traditional methods, the assays defined here eliminated on average 3–4 days from our standard processing time for antiviral testing. Moreover, we were able to utilize human cell lines directly that are not typically amenable to PRNT or plaque assays. The Celigo Image Cytometer can provide an efficient and robust method to rapidly identify potential antiviral drugs to effectively combat the rapidly spreading SARS-CoV-2 virus and its variants during the pandemic.
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Brown, Elizabeth, and Alex Bullock. "Optimisation of seeding of wild-type and FOP fibroblasts for an alkaline phosphatase assay using calcein AM staining." January 22, 2019. https://doi.org/10.5281/zenodo.2546911.
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C2C12 cells did not produce a large fold change in ALP expression following ligand administration (see previous post), therefore we will try to use FOP fibroblasts instead as these have a greater signalling response to BMP ligands. In order to complete this assay I must work out how many cells to seed in order to get a confluent well after 7 days of treatment (the expected period of time required for later compound assays). As the cells were very pale I employed a Calcein AM/Hoechst 3342 stain to mark viable cells.
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Tao, Junyue, Qintao Ge, Jialing Meng, Chaozhao Liang, Zongyao Hao, and Jun Zhou. "Overexpression of DDX49 in prostate cancer is associated with poor prognosis." BMC Urology 23, no. 1 (2023). http://dx.doi.org/10.1186/s12894-023-01251-4.
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Abstract Background There is increasing evidence that DEAD-box helicases (DDX) can act either as promoters or suppressors in various cancer types. Nevertheless, the function of DDX49 in prostate cancer (PCa) is unknown. This study reveals the prognostic and predictive value of DDX49 in PCa. Methods First, we evaluated the expression of DDX49 between PCa and normal tissues based on TCGA and GEO databases. Univariate and multivariate regression analyses were conducted to reveal the risk factors for PCa recurrence. A K–M curve was employed to assess the relationship between DDX49 and recurrence-free survival. In vitro, DDX49 expression was evaluated in PCa and normal prostate cell lines. Furthermore, we constructed a shDDX49 lentivirus to knock down the expression of DDX49. Celigo® Image Cytometer and MTT assay were performed to analyse cell proliferation in PC-3 cells. Cell cycle distribution was detected with flow cytometry analysis. Apoptosis affected by the lack of DDX49 was metred with the PathScan® Stress and Apoptosis Signalling Antibody Array Kit. Results This study shows a high increase in DDX49 in PCa tissues in comparison with normal tissues and that increased DDX49 indicates a poor prognosis among PCa patients. Meanwhile, DDX49 knockdown suppressed the proliferation and migration of PC-3 cells, causing cell cycle arrest in the G1 phase. Stress and apoptosis pathway analysis revealed that the phosphorylation of HSP27, p53, and SAPK/JNK was reduced in the DDX49 knockdown group compared with the control group. Conclusions In summary, these results suggest that high expression of DDX49 predicts a poor prognosis among PCa patients. Downregulation of DDX49 can suppress cell proliferation, block the cell cycle, and facilitate cell apoptosis. Therefore, knockdown of DDX49 is a promising novel therapy for treating patients with PCa.
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Hu, Ting-ting, Jia-wen Yang, Ye Yan, et al. "Detection of genes responsible for cetuximab sensitization in colorectal cancer cells using CRISPR-Cas9." Bioscience Reports 40, no. 10 (2020). http://dx.doi.org/10.1042/bsr20201125.
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Abstract Colorectal cancer (CRC) is a common malignant tumor in digestive tract with highly invasive and metastatic capacity. Drug sensitivity remains a significant obstacle to successful chemotherapy in CRC patients. The present study aimed to explore genes related to cetuximab (CTX) sensitivity in CRC by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Celigo image cytometer was used to detect suitable cells and optimal dosage of CTX. Inhibition rate of CTX on Caco-2 cells was evaluated by cell counting kit-8 (CCK-8) method before and after transfection. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was performed to explore suitable concentration of puromycin and multiplicity of infection (MOI). CRISPR-Cas9, sequencing data quality analysis and cell viability test were used for the selection of genes related to CTX sensitivity in CRC cells. Finally, the selected genes associated with CTX sensitivity in CRC cells were further validated by colony formation and CCK-8 assays. In the present study, Caco-2 cells had a better prolificacy, and CTX 100 μg/ml exhibited a good inhibition trend on the 7th and 14th days of infection. MTT assay indicated that the minimum lethal concentration of puromycin was 2.5 μg/ml. Forty-six candidate genes were preliminarily screened via sequencing data quality analysis. Subsequently, we found that knockout of any of the four genes (MMP15, MRPL48, CALN1 and HADHB) could enhance CTX sensitivity in Caco-2 cells, which was further confirmed by colony formation assay. In summary, MMP15, MRPL48, CALN1 and HADHB genes are related to the mediation of CTX sensitivity in CRC.
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Jin, Lu, Yibin Zhou, Guangqiang Chen, et al. "EZH2-TROAP Pathway Promotes Prostate Cancer Progression Via TWIST Signals." Frontiers in Oncology 10 (February 22, 2021). http://dx.doi.org/10.3389/fonc.2020.592239.
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Trophinin-associated protein (TROAP) has been shown to be overexpressed and promotes tumor progression in some tumors. We performed this study to assess the biological and clinical significance of TROAP in prostate cancer. We downloaded TROAP mRNA expression data from TCGA and GEO databases. We analyzed expressions of TROAP and other genes in prostate cancer tumors at different stages and assessed Gleason scores. We used Celigo image, Transwell, and rescue assays, and flow cytometry detection to assess growth, apoptosis, proliferation, migration, and invasion of the prostate cancer cells. We identified and validated up- and down-stream genes in the TROAP pathway. The mRNA data suggested that TROAP expression was markedly upregulated in prostate cancer compared with its expression in normal tissues, especially in cancers with high stages and Gleason scores. Moreover, a high TROAP expression was associated with poor patient survival. Results of our in vitro assay showed that TROAP knockdown inhibited DU145 and PC3 cell proliferation and viability via cell apoptosis and S phase cycle arrest. The Transwell assay showed that TROAP knockdown inhibited cell migration and invasion, probably through MMP-9 and E-Cadherin modulation. Overexpression of TWIST partially abrogated the inhibitory effects of TROAP knockdown on prostate cancer cells. Our integrative mechanism dissection revealed that TROAP is in a pathway downstream of EZH2 and that it activates the TWIST/c-Myc pathway to regulate prostate cancer progression. In all, we identified TROAP as a driver of prostate cancer development and progression, providing a novel target for prostate cancer treatments.