We thank Raymond Briones and Wen H. Shen (Weill Cornell Medical College, New York, NY, USA) for their help with the development of this protocol. This work has been supported by a Transformative Breast Cancer Consortium Grant from the US DoD BCRP (#W81XWH2120034, PI: Formenti).

The reagents and equipment used in the study are listed in the Table of Materials.
1. Organoid culture
NOTE: TNBC#1 PDTOs were established in our lab based on tumor tissue surgically removed from a patient with triple-negative breast cancer (TNBC) who provided informed consent to participate in a biobanking protocol (IRB21-06023682). After validation by histology and RNA sequencing (RNAseq), TNBC#1 PDTOs are cultured in 66% matrigel drops (so-called &#39;domes&#39;) in enriched DMEM/F12 culture medium (Table 1) and passaged every 2-3 weeks at a 1:2-1:10 ratio28.
<ol>
	<li>Seed PDTOs at a 1:2-1:10 ratio about 2 weeks before irradiation in separate, non-adherent 6-well plates, one per each radiation dose tested (plus negative controls, which will be provided by PDTOs not exposed to ionizing radiation).</li>
	<li>Add 3 mL of fresh enriched DMEM/F12 culture medium to the PDTOs every 3-4 days. 
	NOTE: Seeding and culture conditions may require some adaptation to prevent PDTOs from acquiring a very recognizable, ring-like appearance that is indicative of core necrosis under the microscope and to ensure optimal viability at irradiation.</li>
</ol>
2. Radiation
<ol>
	<li>PDTOs are ready for irradiation once they reach the average size (approximately 100 &#181;m, as preliminary determined under bright field microscopy with a scale reference) and dome occupancy (approximately 80%). Passage the PDTOs at this stage to avoid overcrowding.</li>
	<li>Radiate the PDTO-containing domes using the Small Animal Research Radition Platform (SARRP) irradiator (or an alternative irradiator for in vitro or in vivo applications) in open field mode (no collimator), delivering single doses of 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy. Non-irradiated PDTOs provide appropriate negative control conditions. 
	NOTE: Dosimetry testing calibration should be performed prior to each experiment to ensure that Matrigel-enclosed PDTOs receive the planned radiation dose. These doses were chosen based on preliminary assessments of the radiosensitivity of TNBC#1 PDTOs and, hence, may require adjustments for other PDTOs.</li>
</ol>
3. Dissociation
<ol>
	<li>Immediately after irradiation, wash each PDTO-containing matrigel dome with 2 mL of enriched DMEM/F12 culture medium and resuspend them in 1-3 mL of TrypL-E using a p1000 micropipette. Repeat about 30 times for each well.</li>
	<li>Collect the cell suspension in a 50 mL centrifuge tube and incubate for ~5 min in a water bath at 37 &#176;C.</li>
	<li>Pellet the cells by centrifuging them at 800 x g for 5 min at room temperature.</li>
	<li>Remove excess TrypL-E&#160;to leave ~1 mL in the tube and add 3x the original enzyme volume (see step 3.1) of enriched DMEM/F12 medium to wash the cells.</li>
	<li>Filter the cell suspension using a 40 &#181;m mesh filter. This step ensures that no cell clusters remain at plating and that each new PDTO originates from a single cell.</li>
	<li>Count the cells in an automated cell counter upon staining with 10% trypan blue in PBS, and resuspend them to have 800 cells in 50 &#181;L of enriched DMEM/F12 medium plus 66% matrigel at 4 &#176;C (conditions for 1 dome).</li>
	<li>Plate each dome in an individual well from a live imaging-compatible 48-well culture plate while avoiding the formation of bubbles. Technical triplicates for each radiation dose are recommended.</li>
	<li>Place the plates in a cell culture incubator (37 &#176;C, 5% CO2) for 30 min to solidify the matrigel.</li>
	<li>Add 0.5-1 mL of enriched DMEM/F12 medium, remove potential bubbles, and place plates in the live imaging system inside a cell culture incubator. Let the plate equilibrate to the set temperature for about 30 min before imaging. 
	NOTE: Matrigel solidifies at temperatures &#62;4 &#176;C, implying that this temperature should be preserved until solidification is required.</li>
</ol>
4. Live imaging
<ol>
	<li>On the Incucyte software, select Schedule to Aquire.</li>
	<li>Click on the plus sign in the upper left corner of Launch Add Vessel.</li>
	<li>To scan repeatedly or once, select Scan on Schedule, and then click on Next.</li>
	<li>To create or restore vessel, select Create Vessel New, and then click on Next.</li>
	<li>To select the scan Type, select Organoid, and then click on Next.</li>
	<li>Scan settings: Choose QC &#60; &#39; Phase + Brightfield Image Channels. &#39;4x Objective&#39; is automatically selected. Then click on Next.</li>
	<li>Vessel Selection: Choose the appropriate software-validated plate according to the catalog number.</li>
	<li>Vessel Location: Select the location of the plate on the instrument according to the layout, and then click on Next.</li>
	<li>Scan Pattern: Select wells according to the plate layout, then click on Next.</li>
	<li>Vessel Notebook: Name plate (Optional: name Cell Type and Passage). Create a plate Map by clicking on the plus sign in the upper right corner of the plate layout, &#39;Create plate map&#39;.</li>
	<li>In the Plate Map Editor, indicate cell type by clicking on the Create New Cell icon in the Cells section, apply to appropriate wells by clicking on the plus icon, and select the appropriate wells. Optional: The number of seeded cells and passages can be indicated. Repeat for each cell line.</li>
	<li>Indicate the treatment by clicking on the Create New Cell icon in the Growth Conditions section, apply to appropriate wells by clicking on the plus icon, and select the appropriate wells. Repeat for each experimental condition. Next, click on Okay &#62;&#160;Next.</li>
	<li>Analysis Setup: &#39;Defer analysis until later&#39; is automatically selected; click on Next.</li>
	<li>Scan Schedule: Choose to scan twice a day, indefinitely. Click on Next. Monitor the darkness of control PDTOs for ideal endpoint, as they tend to collapse when overgrown.</li>
	<li>Summary: Check the summary and click on Add to Schedule. 
	NOTE: Scan properties can be visualized, and the schedule can be edited if needed by right-clicking on the scanning timeline.</li>
</ol>
5. Analysis
<ol>
	<li>On the compatible software, select View. This can also be operated from a computer connected to the system via intranet.</li>
	<li>Select the desired experiment by double-clicking on it.</li>
	<li>Select the Launch Analysis icon.</li>
	<li>Select the Create New Analysis Definition, and then click on Next.</li>
	<li>Analysis Type: Select Organoid, and click on Next.</li>
	<li>Image Channels: &#39;Phase + Brightfield&#39; is selected by default, click &#39;Next&#39;.</li>
	<li>Select a set of representative images for analysis. Click on Next. It is recommended to choose early and late time points and different treatment conditions, e.g., control and different radiation doses.</li>
	<li>Perform Analysis Definition for TNBC#1 following the steps below:
	<ol>
		<li>On image: Select Preview Current or &#39;Preview All&#39; to visualize the default mask of current or selected images. Repeat as analysis parameters are changed to update the mask.</li>
		<li>Segmentation: Set the Radius to 300, Sensitivity Background to 80, Edge Split to ON, and Edge Sensitivity to 70.</li>
		<li>Cleanup: Set Hole Fill to 5E+05 &#181;m2 and Adjust Size to 0 pixels.</li>
		<li>Filters: Set no min or max Area, min Eccentricity set to 0.19, and no max Eccentricity. Click on Next.</li>
	</ol>
	</li>
	<li>Scan times and wells: Select all desired scan time points and wells. Click on Next. Optional: Select Analyze Future Scans.</li>
	<li>Save and Apply Analysis Definition: Enter the Definition Name, and click on Next.</li>
	<li>Visualize the Summary, and click on Finish.</li>
	<li>A pop-up window appears to inform the user that the analysis has entered the Analysis Queue. Click on Okay. Once the Analysis Queue window opens, the remaining tasks can be visualized.</li>
	<li>If necessary, retrieve the completed analysis in View by selecting the blue arrow in the Analyses column, next to the Vessel Name, and right-clicking on the Analysis Definition Name. Next, either visualize the raw data on the software by clicking on Graph analysis Metrics, or export for further analysis by selecting Export Analysis Definition. 
	NOTE: Raw data exportation can be customized in the Graph analysis Metrics window. Organoid Count, Total Area, Average Eccentricity, and Darkness can be separately exported, and data organized in the Select Grouping roll-down menu by clicking on None, Columns, Rows or plate Map Replicates. For TNBC#1, for raw data exportation, Graph Analysis Metrics, and for the Select Grouping roll-down option, the option None was chosen. These import parameters were chosen based on preliminary assessments with TNBC#1 PDTOs. They hence may require adjustments for other PDTOs, which is possible on the software based on the &#34;Preview&#34; features. We recommend adapting import parameters on (1) early images that mostly contain single cells or small PDTOs, and (2) images collected at late time points to ensure that large PDTOs are also acquired in both control and treated conditions.</li>
</ol>

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

<ol>
	<li>De Ruysscher, D., 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=Radiotherapy+toxicity.">Radiotherapy toxicity.</a> Nat Rev Dis Primers. 5 (1), 13 (2019).</li><li>Bates, J. E., Sanders, T., Arnone, A., Elmore, S. N. C., Royce, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+density+of+linear+accelerators+and+receipt+of+radiation+therapy+for+prostate+cancer.">Geographic density of linear accelerators and receipt of radiation therapy for prostate cancer.</a> Int J Radiat Oncol Biol Phys. 111, e351-e352 (2021).</li><li>Harmenberg, U., Hamdy, F. C., Widmark, A., Lennern&#228;s, B., Nilsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curative+radiation+therapy+in+prostate+cancer.">Curative radiation therapy in prostate cancer.</a> Acta Oncol. 50, 98-103 (2011).</li><li>Nakano, T., Ohno, T., Ishikawa, H., Suzuki, Y., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advancement+in+radiation+therapy+for+uterine+cervical+cancer.">Current advancement in radiation therapy for uterine cervical cancer.</a> J Radiat Res. 51 (1), 1-8 (2010).</li><li>Spencer, K., Parrish, R., Barton, R., Henry, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palliative+radiotherapy.">Palliative radiotherapy.</a> BMJ. 360, k821 (2018).</li><li>Price, J. M., Prabhakaran, A., West, C. M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+tumour+radiosensitivity+to+deliver+precision+radiotherapy.">Predicting tumour radiosensitivity to deliver precision radiotherapy.</a> Nat Rev Clin Oncol. 20 (2), 83-98 (2023).</li><li>McLaughlin, 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=Inflammatory+microenvironment+remodelling+by+tumour+cells+after+radiotherapy.">Inflammatory microenvironment remodelling by tumour cells after radiotherapy.</a> Nat Rev Cancer. 20 (4), 203-217 (2020).</li><li>Rodriguez-Ruiz, M. E., Vitale, I., Harrington, K. J., Melero, I., Galluzzi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+impact+of+cell+death+signaling+driven+by+radiation+on+the+tumor+microenvironment.">Immunological impact of cell death signaling driven by radiation on the tumor microenvironment.</a> Nat Immunol. 21 (2), 120-134 (2020).</li><li>Cytlak, U. 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=Immunomodulation+by+radiotherapy+in+tumour+control+and+normal+tissue+toxicity.">Immunomodulation by radiotherapy in tumour control and normal tissue toxicity.</a> Nat Rev Immunol. 22 (2), 124-138 (2022).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+Cancer:+New+Dimensions.">Hallmarks of Cancer: New Dimensions.</a> Cancer Discov. 12 (1), 31-46 (2022).</li><li>Groelly, F. J., Fawkes, M., Dagg, R. A., Blackford, A. N., Tarsounas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+damage+response+pathways+in+cancer.">Targeting DNA damage response pathways in cancer.</a> Nat Rev Cancer. 23 (2), 78-94 (2023).</li><li>Renaudin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+DNA+damage+response+in+cancer.">Reactive oxygen species and DNA damage response in cancer.</a> Int Rev Cell Mol Biol. 364, 139-161 (2021).</li><li>Klapp, 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=The+DNA+damage+response+and+inflammation+in+cancer.">The DNA damage response and inflammation in cancer.</a> Cancer Discov. 13 (7), 1521-1545 (2023).</li><li>Constanzo, J., Garcia-Prada, C. D., Pouget, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+assay+to+measure+bystander+cytotoxicity+of+targeted+alpha-particle+therapy.">Clonogenic assay to measure bystander cytotoxicity of targeted alpha-particle therapy.</a> Methods Cell Biol. 174, 137-149 (2023).</li><li>Serrano-Mendioroz, I., Garate-Soraluze, E., Rodriguez-Ruiz, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+assess+clonogenic+survival+of+irradiated+cancer+cells.">A simple method to assess clonogenic survival of irradiated cancer cells.</a> Methods Cell Biol. 174, 127-136 (2023).</li><li>Petroni, 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=Radiotherapy+delivered+before+CDK4/6+inhibitors+mediates+superior+therapeutic+effects+in+ER(+)+breast+cancer.">Radiotherapy delivered before CDK4/6 inhibitors mediates superior therapeutic effects in ER(+) breast cancer.</a> Clin Cancer Res. 27 (7), 1855-1863 (2021).</li><li>Alvarez-Abril, 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=Cytofluorometric+assessment+of+acute+cell+death+responses+driven+by+radiation+therapy.">Cytofluorometric assessment of acute cell death responses driven by radiation therapy.</a> Methods Cell Biol. 172, 17-36 (2022).</li><li>Bodey, R. K., Evans, P. M., Flux, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+linear-quadratic+model+to+combined+modality+radiotherapy.">Application of the linear-quadratic model to combined modality radiotherapy.</a> Int J Radiat Oncol Biol Phys. 59 (1), 228-241 (2004).</li><li>Unkel, S., Belka, C., Lauber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+analysis+of+clonogenic+survival+data:+Statistical+alternatives+to+the+linear-quadratic+model.">On the analysis of clonogenic survival data: Statistical alternatives to the linear-quadratic model.</a> Radiat Oncol. 11, 11 (2016).</li><li>Zitvogel, L., Pitt, J. M., Daill&#232;re, R., Smyth, M. J., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+in+oncoimmunology.">Mouse models in oncoimmunology.</a> Nat Rev Cancer. 16 (12), 759-773 (2016).</li><li>Buqu&#233;, A., Galluzzi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Tumor+immunology+and+immunotherapy+in+mice.">Modeling Tumor immunology and immunotherapy in mice.</a> Trends Cancer. 4 (9), 599-601 (2018).</li><li>Lee, S. Y., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functionally+robust+phenotypic+screen+that+identifies+drug+resistance-associated+genes+using+3D+cell+culture.">A Functionally robust phenotypic screen that identifies drug resistance-associated genes using 3D cell culture.</a> Bio Protoc. 8 (22), e3083 (2018).</li><li>Mina, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bissell:+Context+Matters.">Bissell: Context Matters.</a> Trends Cancer. 1 (1), 6-8 (2015).</li><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>Kim, J., Koo, B. K., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organoids:+Model+systems+for+human+biology+and+medicine.">Human organoids: Model systems for human biology and medicine.</a> Nat Rev Mol Cell Biol. 21 (10), 571-584 (2020).</li><li>van Renterghem, A. W. J., van de Haar, J., Voest, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+precision+oncology+using+patient-derived+assays:+Bridging+genotype+and+phenotype.">Functional precision oncology using patient-derived assays: Bridging genotype and phenotype.</a> Nat Rev Clin Oncol. 20 (5), 305-317 (2023).</li><li>Vitale, I., Manic, G., De Maria, R., Kroemer, G., Galluzzi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+in+stem+cells.">DNA damage in stem cells.</a> Mol Cell. 66 (3), 306-319 (2017).</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>Zhou, Z., 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=Harnessing+3D+in+vitro+systems+to+model+immune+responses+to+solid+tumours:+A+step+towards+improving+and+creating+personalized+immunotherapies.">Harnessing 3D in vitro systems to model immune responses to solid tumours: A step towards improving and creating personalized immunotherapies.</a> Nat Rev Immunol. 24, 18-32 (2024).</li></ol>

Unrelated to this work, SCF is/has been holding research contracts with Merck, Varian, Bristol Myers Squibb, Celldex, Regeneron, Eisai, and Eli-Lilly, and has received consulting/advisory honoraria from Bayer, Bristol Myers Squibb, Varian, Elekta, Regeneron, Eisai, AstraZeneca, MedImmune, Merck US, EMD Serono, Accuray, Boehringer Ingelheim, Roche, Genentech, AstraZeneca, View Ray and Nanobiotix. Unrelated to this work, SD has received consulting/advisory honoraria from Lytix Biopharma, EMD Serono, Ono Pharmaceutical, Genentech, and Johnson &amp; Johnson Enterprise Innovation Inc., and is/has been holding research contracts with Lytix Biopharma, Nanobiotix and Boehringer-Ingelheim. Unrelated to this work, LG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options.

Here, we describe an adaptation of conventional clonogenic assays that harnesses breast cancer PDTOs and live imaging to quantify PDTO radiosensitivity based on (1) the persistence of PTDO-forming stem-like cells upon PDTO irradiation in vitro, and (2) the growth rate of the PDTOs these cells (may) generate. Critical steps of this protocol include (1) the establishment of PDTOs to a dome occupancy enabling good viability, (2) PDTO exposure to ionizing irradiation at different doses, including mock irradiated con...

External beam radiation therapy (RT) is one of the mainstays of modern oncology, reflecting not only a pronounced anticancer activity associated with a well-defined spectrum of generally manageable side effects1, but also an exceptionally widespread clinical availability (most cancer centers in developed countries are equipped with modern linear accelerators for external beam RT)2. In line with this notion, RT is globally employed with success for both curative purposes, generally in the context of early-stage disease3,4, and palliative applications, to contain symptoms (e.g., pain) from metastatic tumors5. That said, not all malignancies are equally sensitive to RT, reflecting a number of cancer cell-intrinsic and microenvironmental features6,7,8,9. As a standalone example, various cancer-associated genetic and epigenetic alterations influencing DNA repair and redox homeostasis have been associated with increased or decreased intrinsic radiosensitivity, largely reflecting the ability of RT to kill cancer cells by causing direct and reactive oxygen species (ROS)-dependent damage to DNA and other macromolecules10,11,12,13.
Over the past decades, clonogenic assays have been routinely employed to assess the radiosensitivity of cultured human and murine cancer cells14,15,16. Indeed, while chemotherapeutics and other stressors often kill malignant cells in a fairly rapid manner, RT employed at clinically relevant doses most often elicit a delayed form of cell death that cannot be simply quantified with short-term flow cytometry- or microscopy-assisted techniques, such as measuring the cellular uptake of vital dyes (which selectively stain dead cells) 24-72 h after exposure to a cytotoxic stimulus17. Besides being straightforward and relying on rather inexpensive reagents, clonogenic assays have been particularly convenient as they allow for the implementation of the so-called &#34;linear quadratic&#34; model, which is a fairly simple mathematical tool for the quantitative analysis of RT dose response18,19. Along similar lines, cultured cancer cells (including established cell lines as well as primary, patient-derived cells) have provided a convenient platform for testing various aspects of cancer biology, including (but not limited to) sensitivity to treatment in a rather straightforward but simplified manner, one major limitation being the absence of a structured tumor microenvironment (TME)20,21. Indeed, two-dimensional cancer cell cultures are intrinsically unable to recapitulate cell-cell communications and interactions with the extracellular matrix and as they occur in cancer22,23. Patient-derived tumor organoids (PDTOs) have emerged as tumor models that at least in part circumvent such limitation24,25,26.
Unfortunately, conventional clonogenic assays cannot be employed to assess the radiosensitivity of PDTOs. On the one hand, irradiating established PDTOs may not necessarily compromise their growth as a multicellular unit, unless their stem-like compartment - which is responsible for the formation and maturation of PDTOs but exhibits increased resistance to DNA damage as compared to more differentiated PDTO-composing cells27- is completely eradicated. On the other hand, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the radiosensitivity of PDTO-composing (including stem-like) cells as exhibited in the context of formed PDTOs. Here, we detail a technique that harnesses live imaging of breast cancer PDTOs to monitor the PDTO-forming efficacy and growth rate of PDTO-derived cells previously exposed to ionizing irradiation compared to their unirradiated counterparts. With required variations largely reflecting the differential biology of individual PDTOs (e.g., culture media requirements, growth rate), we expect this protocol to be suitable for the study of radiosensitivity in a wide panel of PTDOs of both mammary and non-mammary origin.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40 &#181;m mesh filter</td>
			<td>Thomas Scientific</td>
			<td>1164H35</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto T4 Bright Field Cell Counter</td>
			<td>Nexcelom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F/12</td>
			<td>Corning&#160;</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor hEGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Digital Inverted Fluorescence Microscope&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-563-460</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF7</td>
			<td>Peprotech&#160;</td>
			<td>100-19</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Invitrogen&#160;</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Invitrogen&#160;</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>IncuCyte software 2021A</td>
			<td>Sartorius</td>
			<td></td>
			<td>version: 2021A</td>
		</tr>
		<tr>
			<td>Incucyte SX1</td>
			<td>Sartorius</td>
			<td></td>
			<td>model SX1</td>
		</tr>
		<tr>
			<td>Incucyte validated 48 well plate</td>
			<td>Corning&#160;</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Discovery Labware</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>nAc</td>
			<td>Sigma Aldrich&#160;</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td></td>
			<td></td>
			<td>Purchased from the Englander Institute for Precision Medicine, Weill Cornell, NY, USA</td>
		</tr>
		<tr>
			<td>Non-treated 6 well plate</td>
			<td>Cellstar</td>
			<td>657 185</td>
			<td></td>
		</tr>
		<tr>
			<td>NR (Heregulin)</td>
			<td>Peprotech</td>
			<td>100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>p38 MAP inhibitor p38i SB202190</td>
			<td>Sigma Aldrich&#160;</td>
			<td>S7067</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning&#160;</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PenStrep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Rspondin Media</td>
			<td></td>
			<td></td>
			<td>Purchased from the Englander Institute for Precision Medicine, Weill Cornell, NY, USA</td>
		</tr>
		<tr>
			<td>Small Animal Radiation Research Platform (SARRP)&#160;</td>
			<td>Xstrahl Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGFbeta Receptor Inhibitor A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-61</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>112605-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 (RhoKi)</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

Clonogenic assays are routinely employed to measure the radiation sensitivity of cultured human and mouse cancer cells, and they are considered the gold standard for assessing reproductive cell death induced by radiation, which is more relevant to tumor response to radiation therapy than other measures of cell death quantified using short-term techniques. Clonogenic assays were designed for cells that grew in two-dimensional cultures, which do not recapitulate cell-cell interactions and interactions with the cellular matrix as they occur in the tumor. Patient-derived tumor organoids have emerged as tumor models that at least in part circumvent such limitation.Conventional clonogenic assays cannot be employed as such to assess the radiosensitivity of patient-derived tumor organoids. Here, we detail the technique that harnesses live imaging of breast cancer patient-derived tumor organoids to monitor the organoid forming efficacy and growth rate after exposure to ionizing irradiation versus non-irradiated treatment. In the era of personalized medicine, assessing the sensitivity of patient-derived models is of the utmost importance as it provides the information that is required to design and personalize treatment strategies.In the future, we will be addressing the role of the immune cells that are present in the tumor in modulating the response of PDTO to radiations therapy.

TNBC#1 PDTOs were exposed to a single radiation dose of 0 (unirradiated controls), 2 Gy, 4 Gy, 6 Gy, 8 Gy, or 10 Gy on day 0. Immediately thereafter, PDTOs were dissociated to obtain a single-cell suspension for each experimental condition. PDTO-derived cells were next seeded in 48-well plates within 66% matrigel domes (50 &#181;L each) deposited at the center of the wells, in 3 technical replicates per condition. Plates were placed in a live imaging system and imaged every 6 h using the organoid module 4X objective for ...

Read this Scientific Article Protocol about Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids at JoVE.com

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, ...

live-imaging-to-quantify-cellular-radiosensitivity-patient-derived

Research

JoVE Journal

Cancer Research

Irradiation and Processing of Patient-Derived Tumor Organoids for Radiosensitivity Assessment Using Live Imaging

To begin, obtain patient derived tumor organoid or PDTO and add three milliliters of fresh enriched DMEM F/12 culture medium to the PDTO every three to four days. Using the small animal research radiation platform irradiator, irradiate the PDTO containing domes in the open field mode. Immediately after irradiation wash each PDTO containing Nagrigel dome with two milliliters of enriched DMEM F/12 medium.Then using a P 1000 resuspend the PDTO in one to three milliliters of a commercially obtained recombinant enzyme. Transfer the cell suspension into a 50 milliliter centrifuge tube and incubate for five minutes at 37 degrees Celsius. Centrifuge the tube to pellet the cells and aspirate the supernatant to leave approximately one milliliter of recombinant enzyme in the tube.Then wash the cells with enriched DMEM F/12 medium and filter the cell suspension in a 40 micrometer mesh filter to eliminate cell clusters, stain a small sample of the filtered cells with 10%tripin blue in PBS and count them in an automated cell counter. Resuspend the cells to achieve a density of 800 cells in 50 microliters of enriched medium with 66%magrigel. Next plate, each dome in separate wells of an imaging compatible 48 well culture plate and incubate the plate to let the magrigel solidify.Finally, add 0.5 to one milliliter of enriched DMEM F/12 medium to each well for live imaging.

Live Imaging for Quantifying Radiation Therapy Effects on Patient-Derived Tumor Organoids

To begin, obtain the patient-derived tumor organoid, or PDTO, Matrigel Dome and position it in the live imaging system. In the IncuCyte software, select Schedule To Acquire. Click on the plus sign in the upper left corner, select Scan On Schedule, and click Next.Then select Create Vessel New, choose Organoid, followed by QC and Phase plus Brightfield image channels. After choosing the software validated plate, as per the catalog number, select the location of the plate on the instrument and the wells according to the plate layout. To create plate map, click on the plus sign in the upper right corner of the plate layout.Then under the Cells section, click the Add or Edit Cell Type icon and indicate the cell type. Choose the appropriate wells and click on the plus icon. To indicate the treatment, go to growth conditions and click on Create a New Condition.Apply to the appropriate wells with the plus icon. Click on OK, followed by Next. After checking if defer analysis until later is selected, click on Next.Choose to scan twice a day indefinitely. Finally, check the summary and click on Add To Schedule. Irradiation of one of the breast cancer PTDOs in a single dose of two Gray radiation therapy significantly delayed PDTO regrowth, while higher radiation doses completely prevented the regeneration of growing PDTO.

593 Views.  Weill Cornell Medical College.  Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation. 

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

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