We are grateful to the Tissue Procurement Core and Dr. Kristen Coleman at the University of Iowa for providing patient tumor specimens and to Dr. Sofia Gabrilovich in the Department of Obstetrics and Gynecology for assisting with PDO model generation. We also thank Dr. Valerie Salvatico (Agilent, USA) for critical analysis of the manuscript. We acknowledge the following funding sources: NIH/NCI CA263783 and DOD CDMRP CA220729P1 to KWT; Cancer Research UK, Prostate Cancer UK, Prostate Cancer Foundation and Medical Research Council to JSdB. The funders had no role in the design or analysis of experiments or decision to publish.

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

<ol>
	<li>Drost, J., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+cancer+research.">Organoids in cancer research.</a> Nat Rev Cancer. 18 (7), 407-418 (2018).</li><li>Lohmussaar, K., Boretto, M., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-derived+model+systems+in+gynecological+cancer+research.">Human-derived model systems in gynecological cancer research.</a> Trends Cancer. 6 (12), 1031-1043 (2020).</li><li>Sachs, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+living+biobank+of+breast+cancer+organoids+captures+disease+heterogeneity.">A living biobank of breast cancer organoids captures disease heterogeneity.</a> Cell. 172 (1-2), 373-386 (2018).</li><li>de Witte, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+ovarian+cancer+organoids+mimic+clinical+response+and+exhibit+heterogeneous+inter-+and+intrapatient+drug+responses.">Patient-derived ovarian cancer organoids mimic clinical response and exhibit heterogeneous inter- and intrapatient drug responses.</a> Cell Rep. 31 (11), 107762 (2020).</li><li>Adan, A., Kiraz, Y., Baran, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+and+cytotoxicity+assays.">Cell proliferation and cytotoxicity assays.</a> Curr Pharm Biotechnol. 17 (14), 1213-1221 (2016).</li><li>Driehuis, E., Kretzschmar, K., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+patient-derived+cancer+organoids+for+drug-screening+applications.">Establishment of patient-derived cancer organoids for drug-screening applications.</a> Nat Protoc. 15 (10), 3380-3409 (2020).</li><li>Alzeeb, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+cancer+cell+death+analyzed+by+live+cell+imaging+of+spheroids.">Gastric cancer cell death analyzed by live cell imaging of spheroids.</a> Sci Rep. 12 (1), 1488 (2022).</li><li>Deben, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OrBITS:+label-free+and+time-lapse+monitoring+of+patient+derived+organoids+for+advanced+drug+screening.">OrBITS: label-free and time-lapse monitoring of patient derived organoids for advanced drug screening.</a> Cell Oncol (Dordr). 46 (2), 299-314 (2023).</li><li>Tamura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+anticancer+agents+using+patient-derived+tumor+organoids+characteristically+similar+to+source+tissues).">Evaluation of anticancer agents using patient-derived tumor organoids characteristically similar to source tissues).</a> Oncol Rep. 40 (2), 635-646 (2018).</li><li>Le Compte, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+tumor+organoid+drug+screening+using+widefield+live-cell+imaging+for+bulk+and+single-organoid+analysis.">Multiparametric tumor organoid drug screening using widefield live-cell imaging for bulk and single-organoid analysis.</a> J Vis Exp. 190, 64434 (2022).</li><li>Hanson, K. M., Finkelstein, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+accessible+and+high-throughput+strategy+of+continuously+monitoring+apoptosis+by+fluorescent+detection+of+caspase+activation.">An accessible and high-throughput strategy of continuously monitoring apoptosis by fluorescent detection of caspase activation.</a> Anal Biochem. 564-565, 96-101 (2019).</li><li>Isherwood, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+in+vitro+and+in+vivo+imaging+applications:+accelerating+drug+discovery.">Live cell in vitro and in vivo imaging applications: accelerating drug discovery.</a> Pharmaceutics. 3 (2), 141-170 (2011).</li><li>Bi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+patient-derived+organoid+culture+of+gynecologic+cancers+for+disease+modeling+and+drug+sensitivity+testing.">Successful patient-derived organoid culture of gynecologic cancers for disease modeling and drug sensitivity testing.</a> Cancers (Basel). 13 (12), 2901 (2021).</li><li>Binaschi, M., Zunino, F., Capranico, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+action+of+DNA+topoisomerase+inhibitors.">Mechanism of action of DNA topoisomerase inhibitors.</a> Stem Cells. 13 (4), 369-379 (1995).</li><li>Park, Y. Y., Ahn, J. H., Cho, M. G., Lee, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+depletion+during+mitotic+arrest+induces+mitotic+slippage+and+APC/C(Cdh1)-dependent+cyclin+B1+degradation.">ATP depletion during mitotic arrest induces mitotic slippage and APC/C(Cdh1)-dependent cyclin B1 degradation.</a> Exp Mol Med. 50 (4), 1-14 (2018).</li><li>Lukonin, I., Zinner, M., Liberali, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+image-based+phenotypic+chemical+screens.">Organoids in image-based phenotypic chemical screens.</a> Exp Mol Med. 53 (10), 1495-1502 (2021).</li><li>Herpers, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+patient-derived+organoid+screenings+identify+MCLA-158+as+a+therapeutic+EGFR+x+LGR5+bispecific+antibody+with+efficacy+in+epithelial+tumors.">Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR x LGR5 bispecific antibody with efficacy in epithelial tumors.</a> Nat Cancer. 3 (4), 418-436 (2022).</li><li>Ramm, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+live+and+fixed+cell+imaging+method+to+screen+matrigel-embedded+organoids.">High-throughput live and fixed cell imaging method to screen matrigel-embedded organoids.</a> Organoids. 2 (1), 1-19 (2023).</li><li>Dekkers, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+3D+imaging+of+fixed+and+cleared+organoids.">High-resolution 3D imaging of fixed and cleared organoids.</a> Nat Protoc. 14 (6), 1756-1771 (2019).</li><li>Van Hemelryk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+analysis+and+high-content+live-cell+imaging+for+drug+testing+in+prostate+cancer+xenograft-derived+organoids.">Viability analysis and high-content live-cell imaging for drug testing in prostate cancer xenograft-derived organoids.</a> Cells. 12 (10), 1377 (2023).</li><li>Bi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+tyrosine+kinase+anti-angiogenic+cediranib+over+bevacizumab:+Cell+cycle+abrogation+and+synergy+with+chemotherapy.">Advantages of tyrosine kinase anti-angiogenic cediranib over bevacizumab: Cell cycle abrogation and synergy with chemotherapy.</a> Pharmaceuticals (Basel). 14 (7), 682 (2021).</li><li>Bi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+autophagy+overcomes+resistance+to+dual+histone+deacetylase+and+proteasome+inhibition+in+gynecologic+cancer).">Blocking autophagy overcomes resistance to dual histone deacetylase and proteasome inhibition in gynecologic cancer).</a> Cell Death Dis. 13 (1), 59 (2022).</li><li>Guo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H3+as+a+Therapeutic+target+in+advanced+prostate+cancer.">B7-H3 as a Therapeutic target in advanced prostate cancer.</a> Eur Urol. 83 (3), 224-238 (2023).</li><li>Gil, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER3+is+an+actionable+target+in+advanced+prostate+cancer.">HER3 is an actionable target in advanced prostate cancer.</a> Cancer Res. 81 (24), 6207-6218 (2021).</li></ol>

KWT is a co-owner of Immortagen Inc. CJD is an employee of Agilent. JSdB has served on advisory boards and received fees from Amgen, Astra Zeneca, Astellas, Bayer, Bioxcel Therapeutics, Boehringer Ingelheim, Cellcentric, Daiichi, Eisai, Genentech/Roche, Genmab, GSK, Harpoon, ImCheck Therapeutics, Janssen, Merck Serono, Merck Sharp &amp; Dohme, Menarini/Silicon Biosystems, Orion, Pfizer, Qiagen, Sanofi Aventis, Sierra Oncology, Taiho, Terumo, and Vertex Pharmaceuticals; is an employee of the Institute of Cancer Research (ICR), which have received funding or other support for his research work from AZ, Astellas, Bayer, Cellcentric, Daiichi, Genentech, Genmab, GSK, Janssen, Merck Serono, MSD, Menarini/Silicon Biosystems, Orion, Sanofi Aventis, Sierra Oncology, Taiho, Pfizer, and Vertex, and which has a commercial interest in abiraterone, PARP inhibition in DNA repair defective cancers, and PI3K/AKT pathway inhibitors (no personal income); was named as an inventor, with no financial interest for patent 8 822 438, submitted by Janssen that covers the use of abiraterone acetate with corticosteroids; has been the CI/PI of many industry-sponsored clinical trials; and is a National Institute for Health Research (NIHR) Senior Investigator. No other authors have any potential conflicts of interest to disclose.

PDO cultures are becoming an increasingly popular in vitro model system due to their ability to reflect cellular responses and behaviors2. Significant advances have been made in PDO generation, culture, and expansion techniques, yet methods to analyze therapeutic responses have lagged. Commercially available 3D viability kits are lytic endpoint assays, missing out on potentially valuable kinetic response data and limiting subsequent analyses by other methods8. Emerging stud...

Patient-derived tumor organoids (PDOs) are rapidly emerging as a robust model system to study cancer development and therapeutic responses. PDOs are three-dimensional (3D) cell culture systems that recapitulate the complex genomic profile and architecture of the primary tumor1,2. Unlike traditional two-dimensional (2D) cultures of immortalized cancer cell lines, PDOs capture and maintain intratumoral heterogeneity3,4, making them a valuable tool for both mechanistic and translational research. Although PDOs are becoming an increasingly popular model system, commercially available reagents and analysis methods for cellular effects that are compatible with PDO cultures are limited.
The lack of robust methods to analyze subtle changes in treatment response hinders clinical translation. The gold standard cell health reagent in 3D cultures, CellTiter-Glo 3D, utilizes ATP levels as a determinant of cell viability5,6. While this reagent is useful for endpoint assays, there are several caveats, most notably the inability to use samples for other purposes after completion of the assay.
Live-cell imaging is a sophisticated form of kinetic microscopy that, when combined with fluorescent reagents, has the capacity to quantify a variety of cell health readouts within PDO models, including apoptosis7,8,9 and cytotoxicity10. Indeed, live-cell imaging has been integral to high throughput screening of compounds in 2D platforms11,12. Systems such as the Incucyte have made the technology affordable and thus accessible to research groups in a variety of settings. However, the application of these systems to analyze 3D cultures is still in its infancy.
Herein, we describe a method to assess drug response in PDO models of cancer using multiplexed live-cell imaging (Figure 1). Through analysis of bright field images, changes in PDO&#160;size and morphology can be kinetically monitored. Furthermore, cellular processes can be simultaneously quantified over time using fluorescent reagents, such as Annexin V Red Dye for apoptosis and Cytotox Green Dye for cytotoxicity. The methods presented are optimized for the Cytation 5 live-cell imaging system, but this protocol may be adapted across different live-cell imaging platforms.

<table><tbody><tr><td>1.5 mL microcentrfuge tube</td><td>Dot Scientific Inc</td><td>1008113</td><td></td></tr><tr><td>15 mL conical centrifuge tube</td><td>Sarstedt</td><td>62.554.100</td><td></td></tr><tr><td>554 NM LED Cube</td><td>Agilent</td><td>1225012</td><td></td></tr><tr><td>96-well plate</td><td>Corning Costar</td><td>3596</td><td>Prewarmed to 37 &amp;deg;C</td></tr><tr><td>96-well plate</td><td>Agilent</td><td>204626-100</td><td>Prewarmed to 37 &amp;deg;C</td></tr><tr><td>A83-01</td><td>Tocris</td><td>2939</td><td>Final concentration is 500 nM (component of organoid culture media)</td></tr><tr><td>Advanced DMEM/F-12</td><td>Gibco</td><td>12634-010</td><td>component of organoid culture media</td></tr><tr><td>B27 Supplement</td><td>Gibco</td><td>17504044</td><td>Final concentration is 1x (component of organoid culture media)</td></tr><tr><td>BioTek BioSpa 8 Automated Incubator</td><td>Agilent</td><td>BIOSPAG-SN</td><td>Tabletop incubator; BioSpa OnDemand scheduling software comunicates with Gen5 to transfer plates between the BioSpa and the Cytation 5 for imaging (this protocol uses version 1.01.10)</td></tr><tr><td>BioTek Cytation 5 Cell Imaging Multimode Reader</td><td>Agilent</td><td>CYT5PW-SN</td><td>Plate reader; Gen5 software is used for this device (this protocol uses version 3.12.08)</td></tr><tr><td>Cultrex UltiMatrix Reduced Growth Factor Basement Membrane Extract</td><td>R&amp;amp;D Systems</td><td>BME001-10</td><td></td></tr><tr><td>Daunorubicin HCl</td><td>Sigma-Aldrich</td><td>S3035</td><td>Reconstituted in DMSO</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D2438</td><td></td></tr><tr><td>EDTA (0.5 M)</td><td>Thermo Fisher</td><td>AM9260G</td><td></td></tr><tr><td>Forskolin</td><td>Tocris</td><td>1099</td><td>Final concentration is 10 &amp;micro;M (component of organoid culture media)</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>Final concentration is 1x (component of organoid culture media)</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td>Final concentration is 10 mM (component of organoid culture media)</td></tr><tr><td>Human EGF, Animal-Free Recombinant Protein</td><td>Gibco</td><td>AF-100-15-1MG</td><td>Final concentration is 0.5 ng/mL (component of organoid culture media)</td></tr><tr><td>Human FGF-10 Recombinant Protein</td><td>Gibco</td><td>100-26-1MG</td><td>Final concentration is 10 ng/mL (component of organoid culture media)</td></tr><tr><td>Human R-Spondin 1 Recombinant Protein</td><td>Gibco</td><td>120-38-5UG</td><td>Final concentration is 250 ng/mL (component of organoid culture media)</td></tr><tr><td>Hydrocortisone Stock Solution</td><td>StemCell Technologies</td><td>7926</td><td>Final concentration is 500 ng/mL (component of organoid culture media)</td></tr><tr><td>Imaging Filter Cube- GFP</td><td>Agilent</td><td>1225101</td><td></td></tr><tr><td>Imaging Filter Cube- TRITC</td><td>Agilent</td><td>1225125</td><td></td></tr><tr><td>Imaging LED GFP/CFP</td><td>Agilent</td><td>1225001</td><td></td></tr><tr><td>Incucyte Annexin V Red Dye</td><td>Sartorius</td><td>4641</td><td>Reconstituted in organoid culture media</td></tr><tr><td>Incucyte Cytotox Green Dye</td><td>Sartorius</td><td>4633</td><td>DMSO solution</td></tr><tr><td>N-Acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>A7250</td><td>Final concentration is 1.25 mM (component of organoid culture media)</td></tr><tr><td>Nexcelom Bioscience ViaStain AOPI Staining Solution</td><td>Fisher-Scientific</td><td>13366169</td><td>Add 1:50 volume</td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636</td><td>Final concentration is 10 mM (component of organoid culture media)</td></tr><tr><td>Noggin</td><td>R&amp;amp;D Systems</td><td>6057-NG</td><td>Final concentration is 100 ng/mL (component of organoid culture media)</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td>Final concentration is 10 units/mL (component of organoid culture media)</td></tr><tr><td>Phosphate Buffered Saline (1x)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>ant-pm-05</td><td>Final concentration is 100 &amp;micro;g/mL (component of organoid culture media)</td></tr><tr><td>Recombinant Human Heregulin&amp;beta;-1</td><td>Pepro Tech</td><td>100-03</td><td>Final concentration is 37.5 ng/mL (component of organoid culture media)</td></tr><tr><td>Staurosporine solution from Streptomyces sp.</td><td>Sigma-Aldrich</td><td>S6942</td><td></td></tr><tr><td>TrypLE Express</td><td>Life Technologies</td><td>12604013</td><td></td></tr><tr><td>Y-27632, CAS 331752-47-7</td><td>Sigma-Aldrich</td><td>688000</td><td>Final concentration is 5 &amp;micro;M (component of organoid culture media)</td></tr><tr><td>&amp;beta;-Estradiol</td><td>Sigma-Aldrich</td><td>E2758</td><td>Final concentration is 100 nM (component of organoid culture media)</td></tr></tbody></table>

multiplexed live-cell imaging for drug responses in patient-derived organoid models of cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Our objective was to demonstrate the feasibility of using multiplexed live-cell imaging to assess PDO therapeutic response. Proof of concept experiments were performed in two separate PDO models of endometrial cancer: ONC-10817 and ONC-10811 (see Supplementary Figure 1 and Supplementary Figure 2 for ONC-10811 data). Apoptosis (annexin V staining) and cytotoxicity (Cytotox Green uptake) were kinetically monitored in response to the apoptosis-inducing agent, staurosporine. Specifically, PD...

Watch this Scientific Journal Video about Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer at JoVE.com

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

multiplexed-live-cell-imaging-for-drug-responses-patient-derived

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Cancer Research

1.1K Views.  University of Iowa. We passen van patiënten afgeleide organoïdemodellen toe, die zijn gemaakt van echte tumormonsters van patiënten om te onderzoeken hoe de tumoren reageren op medicijnen. We kunnen ook de moleculaire kenmerken definiëren die correleren met respons. Deze resultaten worden vervolgens gebruikt om prioriteit te geven aan de beste nieuwe therapieën om in de toekomst vooruitgang te boeken in de klinische onderzoeken.Een van de grootste uitdagingen bij het gebruik van van patiënten afgele...

Video: Auteur in de schijnwerpers: Ontwikkeling van multiplexed kinetische assays voor organoïde-gebaseerde analyse van geneesmiddelrespons

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Medicine

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Radiation Response in Patient-Derived Tumor Organoids Using a Clonogenic Assay

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Author Spotlight: Radiotherapy and Clonogenic Assays for Advancing Cancer Research and Personalized Medicine

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, ...

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

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