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 d...

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

<ol>
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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. 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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. 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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">
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			<td>40 &#181;m mesh filter</td>
			<td>Thomas Scientific</td>
			<td>1164H35</td>
			<td></td>
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			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
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			<td>Cellometer Auto T4 Bright Field Cell Counter</td>
			<td>Nexcelom</td>
			<td></td>
			<td></td>
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			<td>DMEM F/12</td>
			<td>Corning&#160;</td>
			<td>12634-010</td>
			<td></td>
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			<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>
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			<td>FGF7</td>
			<td>Peprotech&#160;</td>
			<td>100-19</td>
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			<td>GlutaMax</td>
			<td>Invitrogen&#160;</td>
			<td>35050061</td>
			<td></td>
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			<td>Hepes</td>
			<td>Invitrogen&#160;</td>
			<td>15630-080</td>
			<td></td>
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			<td>IncuCyte software 2021A</td>
			<td>Sartorius</td>
			<td></td>
			<td>version: 2021A</td>
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		<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>
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			<td>Matrigel</td>
			<td>Discovery Labware</td>
			<td>354230</td>
			<td></td>
		</tr>
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			<td>nAc</td>
			<td>Sigma Aldrich&#160;</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
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			<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>
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</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