Name: a three-dimensional model of spheroids to study colon cancer stem cells
Uploaded: 2021-01-22T13:00:00
Description: Watch this Scientific Journal Video about A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells at JoVE.com

We acknowledge the imaging and Anipath recherche histology platforms (CRCL, CLB). We are indebted to the pharmacy of the Centre L&#233;on B&#233;rard (CLB) Hospital for the kind gift of FOLFOX and FOLFIRI. We also thank Brigitte Manship for critical reading of the manuscript. The work was supported by the FRM (Equipes FRM 2018, DEQ20181039598) and by the Inca (PLBIO19-289). MVG and LC received support from the FRM and CF received support from ARC foundation and the Centre L&#233;on B&#233;rard.

NOTE: The details of all reagents and materials are listed in the Table of Materials.
1. Spheroid formation
<ol>
	<li>Spheroid culture media
	<ol>
		<li>Prepare basal medium consisting of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 4 mM L-alanyl-L-glutamine dipeptide.</li>
		<li>Prepare DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen/Strep) in basal medium from step 1.1.1.</li>
		<li>Prepare DMEM/basement mem...

<ol>
	<li>Bray, 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=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 68 (6), 394-424 (2018).</li><li>Fearon, E. R., Vogelstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+model+for+colorectal+tumorigenesis.">A genetic model for colorectal tumorigenesis.</a> Cell. 61 (5), 759-767 (1990).</li><li>Rao, C. V., Yamada, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+instability+and+colon+carcinogenesis:+from+the+perspective+of+genes.">Genomic instability and colon carcinogenesis: from the perspective of genes.</a> Frontiers in Oncology. 3, 130 (2013).</li><li>Fearon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+colorectal+cancer.">Molecular genetics of colorectal cancer.</a> Annual Review of Pathology. 6 (1), 479-507 (2011).</li><li>Tran, T. Q., 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=&#945;-Ketoglutarate+attenuates+Wnt+signaling+and+drives+differentiation+in+colorectal+cancer.">&#945;-Ketoglutarate attenuates Wnt signaling and drives differentiation in colorectal cancer.</a> Nature Cancer. 1 (3), 345-358 (2020).</li><li>Batlle, E., 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=Cancer+stem+cells+revisited.">Cancer stem cells revisited.</a> Nature Medicine. 23 (10), 1124-1134 (2017).</li><li>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=The+cancer+stem+cell:+premises,+promises+and+challenges.">The cancer stem cell: premises, promises and challenges.</a> Nature Medicine. 17 (3), 313-319 (2011).</li><li>Barker, 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=Crypt+stem+cells+as+the+cells-of-origin+of+intestinal+cancer.">Crypt stem cells as the cells-of-origin of intestinal cancer.</a> Nature. 457 (7229), 608-611 (2009).</li><li>Dutta, D., Heo, I., 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=Disease+modeling+in+stem+cell-derived+3D+organoid+systems.">Disease modeling in stem cell-derived 3D organoid systems.</a> Trends in Molecular Medicine. 23 (5), 393-410 (2017).</li><li>Bleijs, M., van de Wetering, M., Clevers, H., Drost, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+and+organoid+model+systems+in+cancer+research.">Xenograft and organoid model systems in cancer research.</a> The EMBO Journal. 38 (15), 101654 (2019).</li><li>Kawai, 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=Three-dimensional+culture+models+mimic+colon+cancer+heterogeneity+induced+by+different+microenvironments.">Three-dimensional culture models mimic colon cancer heterogeneity induced by different microenvironments.</a> Scientific Reports. 10 (1), 3156 (2020).</li><li>Ferreira, L. P., Gaspar, V. M., Mano, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+spherically+structured+3D+in+vitro+tumor+models+-Advances+and+prospects.">Design of spherically structured 3D in vitro tumor models -Advances and prospects.</a> Acta Biomaterialia. 75, 11-34 (2018).</li><li>Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid-based+drug+screen:+considerations+and+practical+approach.">Spheroid-based drug screen: considerations and practical approach.</a> Nature Protocols. 4 (3), 309-324 (2009).</li><li>Chaicharoenaudomrung, N., Kunhorm, P., Noisa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+systems+as+an+in+vitro+platform+for+cancer+and+stem+cell+modeling.">Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling.</a> World Journal of Stem Cells. 11 (12), 1065-1083 (2019).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Weiswald, L. -. B., Bellet, D., Dangles-Marie, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spherical+cancer+models+in+tumor+biology.">Spherical cancer models in tumor biology.</a> Neoplasia. 17 (1), 1-15 (2015).</li><li>Nath, S., Devi, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+systems+in+cancer+research:+Focus+on+tumor+spheroid+model.">Three-dimensional culture systems in cancer research: Focus on tumor spheroid model.</a> Pharmacology &amp; Therapeutics. 163, 94-108 (2016).</li><li>Silva-Almeida, C., Ewart, M. -. A., Wilde, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+gastrointestinal+models+and+organoids+to+study+metabolism+in+human+colon+cancer.">3D gastrointestinal models and organoids to study metabolism in human colon cancer.</a> Seminars in Cell &amp; Developmental Biology. 98, 98-104 (2020).</li><li>Chantret, I., Barbat, A., Dussaulx, E., Brattain, M. G., Zweibaum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+polarity,+villin+expression,+and+enterocytic+differentiation+of+cultured+human+colon+carcinoma+cells:+A+survey+of+twenty+cell+lines.">Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: A survey of twenty cell lines.</a> Cancer Research. 48 (7), 1936-1942 (1988).</li><li>Caro, I., 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=Characterisation+of+a+newly+isolated+Caco-2+clone+(TC-7),+as+a+model+of+transport+processes+and+biotransformation+of+drugs.">Characterisation of a newly isolated Caco-2 clone (TC-7), as a model of transport processes and biotransformation of drugs.</a> International Journal of Pharmaceutics. 116 (2), 147-158 (1995).</li><li>Antonchuk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+embryoid+bodies+from+human+pluripotent+stem+cells+using+AggreWellTM+plates.">Formation of embryoid bodies from human pluripotent stem cells using AggreWellTM plates.</a> Methods in Molecular Biology. 946, 523-533 (2013).</li><li>Wolpin, B. M., Mayer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+treatment+of+colorectal+cancer.">Systemic treatment of colorectal cancer.</a> Gastroenterology. 134 (5), 1296-1310 (2008).</li><li>Yaffee, P., Osipov, A., Tan, C., Tuli, R., Hendifar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+systemic+therapies+for+locally+advanced+and+metastatic+rectal+cancer.">Review of systemic therapies for locally advanced and metastatic rectal cancer.</a> Journal of Gastrointestinal Oncology. 6 (2), 185-200 (2015).</li><li>Fujita, K., Kubota, Y., Ishida, H., Sasaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irinotecan,+a+key+chemotherapeutic+drug+for+metastatic+colorectal+cancer.">Irinotecan, a key chemotherapeutic drug for metastatic colorectal cancer.</a> World Journal of Gastroenterology. 21 (43), 12234-12248 (2015).</li><li>Mohelnikova-Duchonova, B., Melichar, B., Soucek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FOLFOX/FOLFIRI+pharmacogenetics:+the+call+for+a+personalized+approach+in+colorectal+cancer+therapy.">FOLFOX/FOLFIRI pharmacogenetics: the call for a personalized approach in colorectal cancer therapy.</a> World Journal of Gastroenterology. 20 (30), 10316-10330 (2014).</li><li>Jordan, N. 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=Impact+of+dual+mTORC1/2+mTOR+kinase+inhibitor+AZD8055+on+acquired+endocrine+resistance+in+breast+cancer+in+vitro.">Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endocrine resistance in breast cancer in vitro.</a> Breast Cancer Research. 16 (1), 12 (2014).</li><li>Mohr, J. 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=The+microwell+control+of+embryoid+body+size+in+order+to+regulate+cardiac+differentiation+of+human+embryonic+stem+cells.">The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells.</a> Biomaterials. 31 (7), 1885-1893 (2010).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+A+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: A complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Luca, A. 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=Impact+of+the+3D+microenvironment+on+phenotype,+gene+expression,+and+EGFR+inhibition+of+colorectal+cancer+cell+lines.">Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.</a> PLoS One. 8 (3), 59689 (2013).</li><li>Petersen, O. W., R&#248;nnov-Jessen, L., Howlett, A. R., 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=Interaction+with+basement+membrane+serves+to+rapidly+distinguish+growth+and+differentiation+pattern+of+normal+and+malignant+human+breast+epithelial+cells.">Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 89 (19), 9064-9068 (1992).</li><li>Nusse, R., 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=Wnt/&#946;-catenin+signaling,+disease,+and+emerging+therapeutic+modalities.">Wnt/&#946;-catenin signaling, disease, and emerging therapeutic modalities.</a> Cell. 169 (6), 985-999 (2017).</li><li>Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A., Zucco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caco-2+cell+line+as+a+model+of+the+intestinal+barrier:+influence+of+cell+and+culture-related+factors+on+Caco-2+cell+functional+characteristics.">The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.</a> Cell Biology and Toxicology. 21 (1), 1-26 (2005).</li><li>Vermeulen, L., Snippert, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+dynamics+in+homeostasis+and+cancer+of+the+intestine.">Stem cell dynamics in homeostasis and cancer of the intestine.</a> Nature Reviews Cancer. 14 (7), 468-480 (2014).</li><li>van der Heijden, M., Vermeulen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+homeostasis+and+cancer+of+the+gut.">Stem cells in homeostasis and cancer of the gut.</a> Molecular Cancer. 18 (1), 66 (2019).</li><li>Barker, N., Bartfeld, S., 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=Tissue-resident+adult+stem+cell+populations+of+rapidly+self-renewing+organs.">Tissue-resident adult stem cell populations of rapidly self-renewing organs.</a> Cell Stem Cell. 7 (6), 656-670 (2010).</li><li>van der Flier, L. G., Haegebarth, A., Stange, D. E., van de Wetering, 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=OLFM4+is+a+robust+marker+for+stem+cells+in+human+intestine+and+marks+a+subset+of+colorectal+cancer+cells.">OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells.</a> Gastroenterology. 137 (1), 15-17 (2009).</li><li>Potten, C. 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=Identification+of+a+putative+intestinal+stem+cell+and+early+lineage+marker;+musashi-1.">Identification of a putative intestinal stem cell and early lineage marker; musashi-1.</a> Differentiation. 71 (1), 28-41 (2003).</li><li>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=The+intestinal+crypt,+a+prototype+stem+cell+compartment.">The intestinal crypt, a prototype stem cell compartment.</a> Cell. 154 (2), 274-284 (2013).</li><li>Clark, D. W., Palle, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenases+in+cancer+stem+cells:+potential+as+therapeutic+targets.">Aldehyde dehydrogenases in cancer stem cells: potential as therapeutic targets.</a> Annals of Translational Medicine. 4 (24), 518 (2016).</li><li>Tomita, H., Tanaka, K., Tanaka, T., Hara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase+1A1+in+stem+cells+and+cancer.">Aldehyde dehydrogenase 1A1 in stem cells and cancer.</a> Oncotarget. 7 (10), 11018-11032 (2016).</li><li>Zoetemelk, M., Rausch, M., Colin, D. J., Dormond, O., Nowak-Sliwinska, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+3D+culture+systems+of+various+complexity+for+treatment+optimization+of+colorectal+carcinoma.">Short-term 3D culture systems of various complexity for treatment optimization of colorectal carcinoma.</a> Scientific Reports. 9 (1), 7103 (2019).</li><li>Garcia-Mayea, Y., Mir, C., Masson, F., Paciucci, R., Leonart, 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=Insights+into+new+mechanisms+and+models+of+cancer+stem+cell+multidrug+resistance.">Insights into new mechanisms and models of cancer stem cell multidrug resistance.</a> Seminars in Cancer Biology. 60, 166-180 (2020).</li><li>Marusyk, A., Janiszewska, M., Polyak, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumor+heterogeneity:+The+Rosetta+Stone+of+therapy+resistance.">Intratumor heterogeneity: The Rosetta Stone of therapy resistance.</a> Cancer Cell. 37 (4), 471-484 (2020).</li></ol>

In vitro 3D models overcome the main experimental drawbacks of 2D cancer cell cultures, as they appear to be more reliable in recapitulating typical tumoral features including microenvironment and cell heterogeneity. Commonly used 3D models of spheroids are scaffold-free (cultured in low-attachment conditions) or scaffold-based (using biomaterials to culture cells). These methods present different disadvantages as they depend on the nature of the scaffold used or give rise to spheroids that are variable in structure and ...

Colorectal cancer (CRC) remains the second leading cause of cancer-associated deaths in the world1. The development of CRC is the result of a progressive acquisition and accumulation of genetic mutations and/or epigenetic alterations2,3, including the activation of oncogenes and inactivation of tumor suppressor genes3,4. Moreover, non-genetic factors (e.g., the microenvironment) can contribute to and promote oncogenic transformation and thus participate in the evolution of CRCs5. Importantly, CRCs are composed of different cell populations, including undifferentiated CSCs and bulk tumor cells displaying some differentiation traits, which constitute a hierarchical structure reminiscent of the organization of the epithelium in a normal colon crypt6,7.
CSCs are considered to be responsible for tumor appearance8, its maintenance and growth, metastatic capacity, and resistance to conventional therapies6,7. Within tumors, cancer cells, including CSCs, display a high level of heterogeneity and complexity in terms of their distinct mutational and epigenetic profiles, morphological and phenotypic differences, gene expression, metabolism, proliferation rates, and metastatic potential9. Therefore, to better understand cancer biology, tumor progression, and acquisition of resistance to therapy and its translation into effective treatments, human preclinical models capturing this cancer heterogeneity and hierarchy are important10,11.
In vitro 2D cancer cell lines have been used for a long time and provide valuable insights into tumor development and the mechanisms underlying the efficacy of therapeutic molecules. However, their limitation with respect to the lack of the phenotypic and genetic heterogeneity found in the original tumors is now widely recognized12. Moreover, nutrients, oxygen, pH gradients, and the tumor microenvironment are not reproduced, the microenvironment being especially important for the maintenance of different cell types including CSCs11,12. To overcome these main drawbacks, several 3D models have been developed to experimentally address and reproduce the complexity and heterogeneity of cancers. In effect, these models recapitulate tumor cellular heterogeneity, cell-cell interactions, and spatial architecture, similar to those observed in vivo12,13,14. Primary tumor organoids established from fresh tumors, as well as cell line-derived spheroids, are largely employed15,16.
Spheroids can be cultured in a scaffold-free or scaffold-based manner to force the cells to form and grow in cell aggregates. Scaffold-free methods are based on the culture of cells under non-adherent conditions (e.g., the hanging-drop method or ultra-low attachment plates), whereas scaffold-based models rely on natural, synthetic, or hybrid biomaterials to culture cells12,13,14. Scaffold-based spheroids present different disadvantages as the final spheroid formation will depend on the nature and composition of the (bio)material used. Although the scaffold-free spheroid methods available so far do not rely on the nature of the substrate, they generate spheroids that vary in structure and size17,18.
This work was aimed at designing a robust and reproducible 3D culture system of spheroids, which are homogenous in size, composed of Caco2 colon adenocarcinoma cells to study CSC biology. Caco2 cells are of particular interest owing to their capacity to differentiate over time19,20, strongly suggesting a stem-like potential. Accordingly, long-term culture of the spheroids revealed the presence of different CSC populations with different responses to chemotherapy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>37 &#181;m Reversible Strainer, Large&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>27250</td>
			<td>To be used with 50 mL conical tubes</td>
		</tr>
		<tr>
			<td>5-Fluorouracil</td>
			<td>Gift from Pharmacy of the Centre Leon Berard (CLB)</td>
			<td>-</td>
			<td>stock solution, 5 mg/100 mL; final concentration, 50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrewell 400 24-well plates</td>
			<td>STEMCELL Technologies</td>
			<td>34411</td>
			<td>1,200 microwells per well for spheroid formation and growth</td>
		</tr>
		<tr>
			<td>Anti Caspase3 - Rabbit</td>
			<td>Cell Signaling</td>
			<td>9661</td>
			<td>dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti Musashi-1 (14H1) - Rat</td>
			<td>eBioscience/Thermo Fisher</td>
			<td>14-9896-82</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution x 100 mL</td>
			<td>STEMCELL Technologies</td>
			<td>07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD133 (13A4) - Rat</td>
			<td>Invitrogen</td>
			<td>14-133-82</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>Anti-CD44 -Rabbit</td>
			<td>Abcam</td>
			<td>ab157107</td>
			<td>dilution 1:2000</td>
		</tr>
		<tr>
			<td>Anti-PCNA - Mouse</td>
			<td>Dako</td>
			<td>M0879</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Anti-&#946;-catenin - Mouse</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7963</td>
			<td>dilution 1:50</td>
		</tr>
		<tr>
			<td>Black multiwell plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>237108</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid Monohydrate</td>
			<td>Sigma</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>CLARIOstar apparatus&#160;</td>
			<td>BMG Labtech</td>
			<td></td>
			<td>microplate reader</td>
		</tr>
		<tr>
			<td>Dako pen</td>
			<td></td>
			<td></td>
			<td>marker pen to mark circles on slides for creating barriers for liquids</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21202</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10037</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10042</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) Glutamax (L-alanyl-L-glutamine dipeptide)</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorogel mounting medium with DAPI</td>
			<td>Interchim</td>
			<td>FP-DT094B</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11077</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td></td>
			<td></td>
			<td>Spheroid image analysis</td>
		</tr>
		<tr>
			<td>Irinotecan&#160;</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 20 mg/mL; final concentration, 100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>iScript reverse transcriptase&#160;</td>
			<td>Bio-Rad</td>
			<td>1708891</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucovorin</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 50 mg/mL; final concentration, 25 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>Nucleospin RNA XS Kit</td>
			<td>Macherey-Nagel</td>
			<td>740902 .250</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxaliplatin</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 100 mg/20 mL;final concentration, 10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR qPCR Premix Ex Taq II (Tli RNaseH Plus)</td>
			<td>Takara</td>
			<td>RR420B</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX- Green</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7020</td>
			<td>nucleic acid stain; dilution 1:5000</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05&#160;%)</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss-Axiovert microscope</td>
			<td></td>
			<td></td>
			<td>inverted microscope for acquiring images of spheroids</td>
		</tr>
	</tbody>
</table>

a three-dimensional model of spheroids to study colon cancer stem cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

As the lack of homogeneity in the size of spheroids is one of the main drawbacks of currently available 3D spheroid culture systems13, the aim of this work was to set up a reliable and reproducible protocol to obtain homogenous spheroids. First, to establish ideal working conditions, different numbers of Caco2 cells were tested, ranging from 50 to 2,000 cells per microwell/spheroid using dedicated plates (Table 1). In effect, each well in these pla...

Watch this Scientific Journal Video about A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells at JoVE.com

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

This new method of spheroid cultures can be used to study the tumorigenicity to analyze the presence of different populations of cancer stem cells and also can be nicely applied to for through high put screen for new drugs. This is a robust and repetitive cultured system for generating large number of homogeneously sized spheroids. Moreover, these spheroids can be used to study cancerous stem cells.This method is set up to study chronic cancer biology but it can be easily set up to study other cancers by changing the 3D culture conditions. Be sure to watch for bubbles in the medium, to not move the plate too much during the spheroids formation and to be careful when harvesting the spheroids into the strainer. Begin by pre treating the wells of a 24 well plate with 500 micro liters of Anti-Adherence Rinsing Solution per well.After a few seconds centrifuge the plate in swinging-bucket rotor and rinse each well with 2 milliliters of warm Basal medium. Observe the plate under the microscope to ensure that the bubbles have been completely removed from the micro wells. If no bubbles are observed, rinse the wells again before adding one milliliter of warm DMEM basement membrane matrix medium to each well.To induce spheroid formation, see the desired concentration of Caco-2 cells in a 2D mono layer in a 10 centimeter dish in DMEM supplemented with 10%FBS and 1%antibiotics. For the culture at 37 degrees Celsius and 5%carbon dioxide in a humidified atmosphere. When an 80%confluency is reached, wash the cells with five milliliters of PBS before treating the culture with two milliliters of Trypsin-EDTA for two to five minutes at 37 degrees Celsius and 5%carbon dioxide.When the cells have detached neutralize the Trypsin with four milliliters of complete DMEM. After counting, collect the cells by centrifugation and resuspend the palette in the appropriate volume of DMEM basement membrane matrix medium to achieve the desired concentration of cells in one milliliter of medium per well. Add one milliliter of cells to each well of the prepaid 24 well plate, followed by the addition of one milliliter of DMEM basement membrane matrix medium to each well.After seeding, immediately centrifuge the plate and use a light microscope to verify that the cells have been evenly distributed across the micro wells. Then place the plate in the cell culture incubator for 48 hours without disturbing the cells. To harvest the spheroids, at the end of the incubation, use a serological pipette and half of the supernatant in each well to gently dislodge the spheroids from their micro wells.When all of the spheroids have been detached use the pipette to carefully transfer the 3D cultures from each well unto a 37 micron reversible strainer on top of a 15 milliliter conical tube. Wash each well three times with one milliliter of prewarmed Basal medium per wash to collect any remaining spheroids and add these additional spheroids to the strainer. After the last wash check the plate under the microscope to confirm that all of the spheroids have been removed from the micro wells.Then invert the strainer over a new 15 milliliter tube and wash the bottom of the strainer with fresh DMEM basement membrane matrix medium to collect the spheroids into the tube. Spheroids are rising from 500 cells demonstrate the most homogeneous increase in size over time. With 500 and 600 cells per spheroid determine to be the optimal number of cells under which good growth and low variability can be achieved.The addition of basement membrane matrix enhances spheroid growth and homogeneity. In long term culture, the cells appear as dense multi layers at day three. While flattened cells arranged in mono layers are clearly visible at day 10.Interestingly, a lumen appears within the spheroids from day five onward. In addition, a clear increase in proliferating cell nuclear antigen positive cells is observed at days five and seven that declines by day 10. Surprisingly, activated Caspace 3 is observed in very few cells at days three and five only.While high levels of Beta-catenin expression are observed at every time point. The potential of the spheroids to differentiate into Anterosites is evidenced by their Alkaline Phosphatase and solute carrier family 2A5 mRNA expression. In addition, this spheroids express typical cancer stem cell markers and respond to combination chemotherapy treatments routinely administered to colorectal cancer patients.So spheroids are composed of different cell populations. They then can be nicely used to analyze cell specifical responses to stimuli such as refractors and eventually for drug responses.

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Research

JoVE Journal

Cancer Research

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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 ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...