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• W2942656692 abstract "Resource29 April 2019free access Transparent process 3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids Theresa E Schnalzger Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany University of Konstanz, Konstanz, Germany Search for more papers by this author Marnix HP de Groot Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany Search for more papers by this author Congcong Zhang Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Mohammed H Mosa Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Search for more papers by this author Birgitta E Michels Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Faculty of Biological Sciences, Goethe University, Frankfurt, Germany Search for more papers by this author Jasmin Röder Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Search for more papers by this author Tahmineh Darvishi Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Winfried S Wels Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Search for more papers by this author Henner F Farin Corresponding Author [email protected] orcid.org/0000-0003-1558-5366 Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Search for more papers by this author Theresa E Schnalzger Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany University of Konstanz, Konstanz, Germany Search for more papers by this author Marnix HP de Groot Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany Search for more papers by this author Congcong Zhang Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Mohammed H Mosa Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Search for more papers by this author Birgitta E Michels Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Faculty of Biological Sciences, Goethe University, Frankfurt, Germany Search for more papers by this author Jasmin Röder Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Search for more papers by this author Tahmineh Darvishi Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Winfried S Wels Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Search for more papers by this author Henner F Farin Corresponding Author [email protected] orcid.org/0000-0003-1558-5366 Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany Search for more papers by this author Author Information Theresa E Schnalzger1,2, Marnix HP Groot1, Congcong Zhang1,3,4, Mohammed H Mosa1,3,4,5, Birgitta E Michels1,3,4,5,6, Jasmin Röder1,5, Tahmineh Darvishi1,3,4, Winfried S Wels1,3,4,5 and Henner F Farin *,1,3,4,5 1Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany 2University of Konstanz, Konstanz, Germany 3German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany 4German Cancer Research Center (DKFZ), Heidelberg, Germany 5Frankfurt Cancer Institute, Goethe University, Frankfurt, Germany 6Faculty of Biological Sciences, Goethe University, Frankfurt, Germany *Corresponding author. Tel: +49 69 63395 520; E-mail: [email protected] EMBO J (2019)38:e100928https://doi.org/10.15252/embj.2018100928 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR-engineered NK-92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen-specific cytotoxicity was studied with CAR-NK-92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor-specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner. Synopsis Establishment of a preclinical in vitro organoid model allows testing the efficacy and safety of solid tumor cancer immunotherapy with chimeric antigen receptor (CAR)-engineered lymphocytes in a tissue-like environment. 3D live-imaging of single colorectal cancer (CRC) organoids is highly sensitive and allows to validate treatment assumptions to minimize therapy side effects. Co-culture of human natural killer (NK) cells with normal or CRC organoids on an ECM layer allows stable effector-target cell interaction. Luciferase-based cytotoxicity assessment is benchmarked with EPCAM-directed effector NK cells. Low expression of EGFRvIII neoantigen is sufficient to render CRC organoids susceptible to lysis. CAR targeting of FRIZZLED receptors in intestinal organoids reveals off-target cytotoxicity against wild-type epithelia of human origin. Introduction To increase the response rates to cancer immunotherapy, immune effector cells can be engineered to recognize tumor-associated antigens (TAA) expressed on the surface of cancer cells. Introduction of chimeric antigen receptors (CAR) has shown impressive success in various hematological malignancies, and the use of autologous CAR-engineered T cells in relapsed B-cell acute lymphoblastic leukemia and relapsed large B-cell non-Hodgkin lymphoma was recently approved by the Food and Drug Administration (June & Sadelain, 2018). However, for solid cancers, CAR-based therapies have faced several challenges: Most commonly, overexpressed tumor antigens show expression also in normal tissues, which may lead to serious side effects (Bonifant et al, 2016). Targeting cancer-specific antigens, such as neoantigens, requires highly individualized approaches, and access to suitable preclinical models to test efficacy and safety. Clinical application of autologous CAR-T cells is both labor- and cost-intensive. The established natural killer (NK) cell line NK-92 has been described as an alternative source for CAR-engineered immune cells. The NK-92 cell line was derived from a non-Hodgkin's lymphoma (Gong et al, 1994) and displayed high non-MHC-restricted cytolytic activity in a number of preclinical studies (Suck et al, 2016). Together with the possibility of robust ex vivo expansion and engineering, NK-92 cells may serve as a standardized platform for off-the-shelf CAR reagents (Zhang et al, 2017). Very recently, safety data from a phase I clinical trial with CD33-specific CAR-NK-92 cells were reported (Tang et al, 2018). Another CAR-NK-92 cell product targeting the human epidermal growth factor receptor 2 (HER2) has entered a phase I clinical trial for the treatment of glioblastoma (NCT03383978; https://clinicaltrials.gov). Metastatic colorectal carcinoma (CRC) accounts for the fourth most common cause of cancer-related deaths worldwide (Ferlay et al, 2015). Conventional treatment options such as radiation and chemotherapy are often associated with severe side effects and disease recurrence. Hence, alternative and more personalized therapeutic strategies are urgently needed. Harnessing the enormous potential of the immune system appears an encouraging option to improve therapy outcomes in CRC patients. For microsatellite instable (MSI) tumors that are strongly immunogenic, checkpoint inhibition has shown a high clinical response rate (Le et al, 2015). However, microsatellite stable (MSS) tumors are refractory to immune checkpoint blockade, and patients with particularly poor prognosis show low leukocyte infiltration or reduced cytotoxic gene expression (Galon et al, 2006; Mlecnik et al, 2016) and may benefit from CAR-based therapies. To develop effective CAR strategies, physiological preclinical models are required that recapitulate the individual tumor phenotype as well as the complex three-dimensional (3D) tissue environment. The organoid culture system first described by Sato et al (2009, 2011) allows long-term ex vivo expansion of gastrointestinal stem cells in a 3D extracellular matrix. The technology has been used to establish living biobanks of cancer and normal tissues that preserve the genetic and functional heterogeneity among CRC patients (van de Wetering et al, 2015; Fujii et al, 2016). Addition of stromal cells further allows to reconstruct the tissue microenvironment and to study immuno-epithelial crosstalk (Farin et al, 2014; Rogoz et al, 2015; Nozaki et al, 2016; Noel et al, 2017). Moreover, tumor-reactive T cells can be selectively expanded in co-culture with tumor organoids from MSI patients (Dijkstra et al, 2018). For quantitative characterization of cytotoxic responses to 3D organoids, there is an unmet need for standardized protocols. Monitoring of cell-mediated killing is classically performed after single cell dispersal (Zaritskaya et al, 2010) that may result in spontaneous lysis due to sensitivity to enzymatic digest. In suspension, effector cell recruitment, motility, and interaction length cannot be addressed, which are key parameters for cytotoxicity in vivo (Weigelin et al, 2015; Halle et al, 2016). Current read-outs for cell-mediated cytotoxicity, such as flow cytometry-based viability assays or enzyme release assays, only allow detection for a limited amount of time, which may not be sufficient to recapitulate the kinetics in a 3D environment. Finally, T-cell responses (e.g., using PBMCs) show inherent donor variability and the directed antigens are unknown in most cases. In order to study cytotoxic responses to specific TAAs, we have taken advantage of the standardized CAR-NK-92 system. The aim was to establish a quantitative platform for CAR-mediated cytotoxicity toward patient-derived colon organoids. Endpoint and dynamic assays were developed to monitor cytotoxic responses in a 3D environment. We demonstrate targeting of universal antigens, neoantigens, and new candidate TAAs providing an informative platform for preclinical screening of efficacy and tumor cell specificity of CAR-based strategies. Results Directing CAR cytotoxicity to human colon organoids To establish a model for CAR targeting of organoids, we first tested the compatibility of NK-92 cells with organoid culture medium. After 24 and 72 h of culture, we observed a comparable expansion as in the regular NK-cell medium (Fig 1A), even in the absence of human serum and IL-2 that are usually required for growth and activity. To test if the NK-92 function is preserved under these conditions, a standard cytotoxicity assay was performed using HER2-specific CAR-NK-92 cells that were co-incubated with MDA-MB453 (HER2-positive) or MDA-MB468 (HER2-negative control) breast carcinoma cells as targets (Schönfeld et al, 2015). Specific lysis was strongly reduced in organoid medium even in the presence of human plasma or IL-2 (Fig 1B). By testing all factors of the organoid medium individually, we identified nicotinamide as responsible for this inhibition (Fig 1C), a non-essential factor as shown by cell viability assays (Fig 1D) that was subsequently omitted from the medium. To identify universal co-culture conditions that allow effective killing of both normal and tumor human colon organoids, we used CAR-NK-92 cells that target EPCAM as a ubiquitously expressed epithelial surface antigen (Trzpis et al, 2007; Sahm et al, 2012; Fig 1E and F). Organoids were either embedded in Matrigel, on a thin layer of Matrigel or in suspension, followed by addition of EPCAM-CAR NK-92 cells or the control parental NK-92 cells. Microscopic inspection showed that NK cells readily migrated on the surface but were incapable to penetrate the dense extracellular matrix (ECM). After 8 h, only culturing on a Matrigel-coated layer but not in suspension resulted in a marked CAR-induced lysis (Fig 1G). Our results suggest that co-culture on an ECM layer can increase NK-cell migration and/or stabilize the effector–target cell interaction and was therefore used for all subsequent experiments. Figure 1. Organoid NK-92 co-culture model for efficient 3D killing assays A. Growth of NK-92 cells in standard NK medium and organoid medium. 3.6 × 104 cells/ml were seeded, and organoid medium was supplemented with IL-2 or human plasma (HP) followed by cell counting after 0, 24, 48, and 72 h. Mean ± SD on log2 scale, in n = 3 parallel cultures. ns: P > 0.05 (unpaired t-test). B. Reduced cytotoxic activity of CAR NK-92 cells in organoid medium. FACS-based cytotoxicity assay using HER2-targeted CAR-NK-92 cells. Target cells were MDA-MB453 (HER2+) and MDA-MB468 (HER2−) breast carcinoma cells. Mean CAR-specific lysis (± SD) was determined in triplicates. Significance was analyzed by unpaired t-test. C. Identification of nicotinamide (nico) as inhibitory factor in a standard cytotoxicity assay with MDA-MB453 target cells. Each component of the organoid medium was added to the NK medium in a standard cytotoxicity assay (as in B). Mean CAR-specific lysis (± SD) was determined in three independent experiments. Significant P-values (compared to control) from unpaired t-test analysis are shown. Nac: N-acetylcysteine; SB: SB202190. D. Unaffected growth of normal colon organoids in nicotinamide-free medium. Mean cell viability (± SD; in n = 3 wells; CellTiter-Glo assay) compared to regular medium. The experiment was repeated twice independently. Scale bars: 500 μm. E, F. Flow cytometric and immunofluorescence microscopy analysis of EPCAM staining in normal colon organoids. Scale bar: 200 μm. D. Co-culture strategies for analysis of CAR-NK-92-mediated cytotoxicity. Organoids were embedded in Matrigel, or seeded on a Matrigel-coated surface, or kept in suspension, followed by addition of EPCAM-CAR NK-92 cells (E:T ratio was 4:1). Schematic representation of experimental conditions (top) and morphological images after 0 and 8 h of co-culture. Red arrows show examples of organoids that are efficiently lysed. In the magnified images (bottom row), abundant apoptotic bodies are present around the lysed organoids. Scale bars: 500 μm. Download figure Download PowerPoint Quantitative analysis of CAR-mediated cytotoxicity using a luciferase-based 3D assay For quantitative monitoring of CAR-mediated cytotoxic activity against 3D organoids, we sought to develop assays that do not require single cell digestion. Firefly luciferase reporter has been described as a convenient read-out of viable cells following cytotoxicity against 2D cell lines (Fu et al, 2010). To adapt this strategy, luciferase/GFP expression was introduced into organoids using lentiviral transduction, which allowed homogenous expression (Fig EV1A and B). By titration of organoids, linear and sensitive cell detection was confirmed (Fig EV1C). In addition, detergent-induced cell lysis caused rapid solubilization of the cellular luciferase activity with < 20% residual signal detected in cellular debris, allowing accurate quantification of viable organoid cells. Click here to expand this figure. Figure EV1. Luciferase activity as a quantitative read-out for lysis-resistant target cells Lentiviral construct to drive co-expression of luciferase 2 and EGFP (separated by a P2A peptide). Stable expression is selectable by the puromycin resistance marker. Additional features are long-terminal repeats (LTR), Rev response element (RRE), cPPT central polypurine tract, and the phosphoglycerate kinase (PGK) promoter. Patient-derived normal and tumor colon organoids were lentivirally transduced to stably co-express luciferase and GFP. Homogenous expression of GFP was observed. Scale bars: 1,000 μm. Bottom: Flow cytometric analysis of GFP expression. GFP-negative organoids are shown as control. Linear detection of viable organoids. Luciferase activity (in relative light units, RLU) was quantified either after complete lysis (black line), or in the supernatant (blue line) or the remaining cell debris (green line) following cell permeabilization. Mean values (± SD) from n = 3 replicates. The coefficients of determination (R2) are displayed. An organoid number of 100% corresponds to 1.25 × 105 epithelial cells. Note that residual signal after lysis was below 20% of total activity. Quantification of luciferase activity in different co-culture settings (as in Fig 1G). Mean luciferase activity (± SD) from n = 3 replicates relative to growth in Matrigel. The presence of EPCAM-CAR NK-92 cells (E:T ratio was 1:1, for 8 h) reduces luciferase signal of normal organoids after seeding on a Matrigel-coated surface. No reduction is detected after organoid seeding in Matrigel or in suspension. The experiment was repeated three times. Significance was analyzed by unpaired t-test. Note that organoids were seeded on Matrigel 24 h before the start of co-culture, which causes reduction of signal compared to standard culture. Download figure Download PowerPoint Using this assay, we tested the kinetics of cytotoxic activity of EPCAM-CAR NK-92 cells toward patient-derived normal and tumor organoids (Fig 2A). Luciferase activity was significantly reduced only after co-culture with organoids on a Matrigel-coated layer, but not with organoids embedded in Matrigel or in suspension (Fig EV1D), confirming our morphological observations above (Fig 1G). After an initial refractory period of 2 h, a steady increase in cytotoxicity could be observed for both parental and CAR-engineered NK-92 cells (Fig 2B). However, target cell lysis was significantly increased in the case of EPCAM-directed effector cells, where after 24 h, ~80% lysis was achieved. CAR-specific lysis was determined by subtraction of cell lysis by parental NK-92 cells and reached ~40% after 12 h. In order to determine the optimal effector-to-target (E:T) ratio, the absolute organoid cell number was first determined in an image-based manner (Appendix Fig S1). Increasing cytotoxic efficiency could be observed at higher E:T ratios (Fig 2C) that were equally effective against normal and tumor organoids (which expressed similar levels of EPCAM). Next, the organoid and NK number was varied under constant E:T ratio to test whether the response is sensitive to the absolute cell densities. We found that the density had no major impact on the efficiency of killing, indicating that CAR cell recruitment can occur even at reduced effector cell density (Fig 2D). Moreover, we could achieve non-perturbed lysis also in a tissue-like environment, where organoids were seeded on a layer of colon fibroblasts, indicating that presence of EPCAM-negative fibroblasts does not interfere with CAR cell recruitment (Fig 2E and F). Together, our results demonstrate that luciferase-based measurement can serve as flexible read-out for cytotoxicity in advanced tumor models. Figure 2. Luciferase-based quantification of CAR-mediated cytotoxicity toward normal and tumor organoids A. Schematic workflow. Luciferase-expressing organoids allow quantification of residual cells after CAR-mediated killing. B. Kinetic analysis of EPCAM-directed CAR-NK-92 cytotoxicity toward normal colon organoids. Remaining luciferase activity, normalized to organoids cultured alone, is shown as “target cell lysis”. Mean values (± SD) from n = 3 independent experiments. E:T ratio was 2:1. Statistical significance was analyzed by unpaired t-test. C. EPCAM-CAR-specific lysis of normal and tumor organoids at different E:T ratios. Mean values (± SD) from n = 3 independent experiments after 8 h. ns: P > 0.05 (unpaired t-test). D. Influence of organoid/CAR cell density. At a constant E:T ratio of 1:1, decreasing numbers of organoids/NK-92 cells were co-cultured. EPCAM-CAR-specific lysis was measured in triplicates after 8 h (mean ± SD), and the experiment was replicated twice. ns: P > 0.05 (unpaired t-test). E, F. EPCAM-CAR targeting of organoids in the presence of human colonic primary fibroblasts. E:T ratio was 1:1. (E) Fluorescence microscopy analysis of luciferase/GFP-expressing organoids (green); anti-CD45-APC-labeled NK-92 cells (magenta) and dsRED-expressing fibroblasts (red) after 8 h. Scale bars: 200 μm. (F) Luciferase-based cytotoxicity assay. Unaffected CAR-specific lysis in the presence and absence of fibroblasts (measured in triplicates; mean ± SD). ns: P > 0.05 (unpaired t-test). Download figure Download PowerPoint Dynamic monitoring of CAR-NK-92 cytotoxicity by live-cell imaging To study cytotoxic responses in real time and at cellular resolution, we set up a confocal live-cell imaging protocol. Colon organoids stably expressing GFP were cultured alone or together with EPCAM-CAR cells or parental NK-92 cells that were labeled by anti-CD45 staining. Spinning-disk microscopy in multiwell format allowed stable 3D imaging during 10 h and at 6-min intervals (Fig 3A). From maximum image projection data, automatic image analysis was performed (see Appendix Fig S2) in organoids cultured alone (Movies EV1 and EV2), together with parental NK-92 cells (Movies EV3 and EV4) or with EPCAM-CAR cells (Movies EV5 and EV6). The loss of fluorescent organoid area over time was quantified as a measure of target cell killing (Figs 3B and C, and EV2A). To confirm a linear detection of target cell area, organoids were seeded at different concentrations and analyzed before and 8 h after addition of CAR or parental NK-92 cells (Fig EV2B). Average GFP+ area from multiple imaging positions showed a significant and progressive decline after addition of EPCAM-specific NK-92 but not parental NK-92 cells or organoids alone (Fig 3D). CAR-specific lysis was evident after 2 h, reaching a level of ~40% after 10 h. Side-by-side comparison of image- and luciferase-based read-out showed very similar results, thus corroborating our protocols (Fig 3E). To study NK-92 recruitment to the organoids, a second image analysis workflow was established that allows time-resolved measurement of NK density in an area extending 50 μm around each organoid (see Appendix Fig S2). We detected a progressive recruitment of CAR NK-92 cells that reached saturation after 2 h (Fig 3F), paralleling the observed kinetics of lysis. Parental NK-92 cells were also enriched to the organoids compared to the overall density, albeit at a lower level than CAR cells (Fig 3F). These results suggest that parental NK-92 cells do not achieve sufficiently high concentration or stable interaction with target cells to exert damage. To address if the organoid size has an impact on the killing efficiency, organoids were categorized by their initial size into three groups followed by tracking on a single organoid level (Fig EV2C–E). Comparison of the rate of relative area loss showed that small organoids are more effectively attacked (Fig EV2F), while larger organoids require longer time periods for lysis. For larger organoids, a stronger correlation between average of CAR-NK density and relative area loss was found, indicating that a prolonged period of high local effector density is required to achieve complete lysis (Fig 3G). Figure 3. Continuous imaging-based monitoring of cytotoxic activity toward organoids Live imaging strategy using spinning-disk microscopy. Exemplary data with GFP-expressing organoids (green) and anti-CD45-APC-labeled NK-92 cells (magenta) at 0 and 10 h of co-culture. Maximum intensity projections are shown. Organoid outlines are automatically detected; white boxes indicate organoids tracked in (C). Scale bars: 200 μm. See also Fig EV2 and Movies EV1, EV2 EV3 EV4 EV5 and EV6. Single organoid tracking (area) during CAR-mediated killing. Quantitative monitoring of cytotoxicity by loss of GFP+ organoid area. Mean values from n = 4 imaging positions (± SD) are shown relative to the area detected at t = 0. Slopes were compared by linear regression; statistically significant differences were observed (ANCOVA). Comparative kinetics of CAR-specific lysis detected by image-based analysis (black line) and luciferase-based measurement (green line; data are from Fig 2B). Time-resolved recruitment of NK-92 cells to target organoids. NK-cell density was automatically determined in a 50 μm surrounding region (see Appendix Fig S4A). Mean ± SD from n = 4 imaging positions. Hatched lines show the NK-92 density on the entire image. Slopes were compared by linear regression; significant differences were observed (ANCOVA). Correlation of area loss and NK-cell density on a single organoid level. Organoids were classified as small (area < 2,000 μm2; n = 12) and large (area > 2,000 μm2; n = 13), respectively. Maximum relative area loss and average NK density for the entire co-culture period were plotted, and coefficients of determination (R2) are shown. Note that large organoids contribute mostly to overall recruitment in (F). Data information: All experiments were performed at an E:T ratio of 2:1. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Imaging-based quantification and tracking of individual organoids Kinetic imaging data of single organoids (see Fig 3B and C). GFP-positive organoids cultured alone or with parental NK-92 cells or EPCAM-CAR cells. NK-92 cells were labeled with anti-CD45-APC. Maximum intensity projections of merged channels and the single GFP channel are shown. Organoid areas detected by the analysis software and expanded areas for determining NK-cell recruitment are marked in color. Scale bars: 100 μm. Linear detection of organoid area. Organoids were seeded on Matrigel layer at different concentrations and co-cultured with parental NK-92 cells or EPCAM-CAR cells. For each concentration, the organoid area was automatically quantified in 110 imaging fields before (0 h) or after (8 h) addition of NK-92 cells. The entire imaging region was subdivided into n" @default.
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• W2942656692 date "2019-04-29" @default.
• W2942656692 modified "2023-10-14" @default.
• W2942656692 title "3D model for <scp>CAR</scp> ‐mediated cytotoxicity using patient‐derived colorectal cancer organoids" @default.
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• W2942656692 doi "https://doi.org/10.15252/embj.2018100928" @default.
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