<meta charset="utf8"></head><body>使用克隆形成测定法检测患者来源的肿瘤类器官的放射反应

<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>

<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>
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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>
		</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

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.

<meta charset="utf8"></head><body>作者聚焦：用于推进癌症研究和个性化医疗的放射治疗和克隆形成检测

实时成像量化患者来源的肿瘤类器官中的细胞放射敏感性

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.

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

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

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JoVE Journal

Cancer Research

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

590 次观看.  Weill Cornell Medical College. 克隆形成测定通常用于测量培养的人和小鼠癌细胞的辐射敏感性，它们被认为是评估辐射诱导的生殖细胞死亡的金标准，与使用短期技术量化的其他细胞死亡指标相比，这与肿瘤对放射治疗的反应更相关。克隆形成测定法是为在二维培养物中生长的细胞设计的，这些细胞不会概括细胞间相互作用以及与细胞基质的相互作用，因为它们发生在肿瘤中。?...