Name: discovery of driver genes in colorectal ht29-derived cancer stem-like tumorspheres
Uploaded: 2020-07-22T13:00:00
Description: Watch this Scientific Journal Video about Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres at JoVE.com

The authors thank the Radiation Biology Core Laboratory of Institute for Radiological Research, Chang Gung Memorial Hospital, for technical support. This study was supported by grants from Chang Gung Memorial hospital (CMRPD1J0321),&#160;Cheng Hsin General Hospital (CHGH 106-06), and Mackay Memorial Hospital (MMH-CT-10605 and MMH-106-61). Funding bodies did not have any influence in the design of the study and data collection, analysis and interpretation of data or in writing the manuscript.

1. Cell culture and tumorsphere formation
<ol>
	<li>Culture HT29 cells in a 10 cm dish containing Dulbecco&#8217;s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic (P/S).</li>
	<li>Grow the cells in an incubator at 37 &#176;C with 5% CO2 and 95% humidity under aseptic conditions, until they reach 80% confluency.</li>
	<li>Trypsinize HT29 cells with 1 mL of 0.25% trypsin for 5 min at 37 &#176;C and consequently neutralize the trypsin by adding 2 mL of DM...

<ol>
	<li>Rawla, P., Sunkara, T., Barsouk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+colorectal+cancer:+incidence,+mortality,+survival,+and+risk+factors.">Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors.</a> Przegl&#261;d Gastroenterologiczny. 14 (2), 89-103 (2019).</li><li>Wong, M. C., Ding, H., Wang, J., Chan, P. S., Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+risk+factors+of+colorectal+cancer+in+Asia.">Prevalence and risk factors of colorectal cancer in Asia.</a> Intestinal Research. 17 (3), 317-329 (2019).</li><li>Liu, 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=Positive+expression+of+basic+transcription+factor+3+predicts+poor+survival+of+colorectal+cancer+patients:+possible+mechanisms+involved.">Positive expression of basic transcription factor 3 predicts poor survival of colorectal cancer patients: possible mechanisms involved.</a> Cell Death &amp; Disease. 10 (7), 509 (2019).</li><li>Slattery, M. L., 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=Dysregulated+genes+and+miRNAs+in+the+apoptosis+pathway+in+colorectal+cancer+patients.">Dysregulated genes and miRNAs in the apoptosis pathway in colorectal cancer patients.</a> Apoptosis. 23 (3-4), 237-250 (2018).</li><li>Arteaga, C. L., Engelman, 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=ERBB+receptors:+from+oncogene+discovery+to+basic+science+to+mechanism-based+cancer+therapeutics.">ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics.</a> Cancer Cell. 25 (3), 282-303 (2014).</li><li>Yarden, Y., Pines, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ERBB+network:+at+last,+cancer+therapy+meets+systems+biology.">The ERBB network: at last, cancer therapy meets systems biology.</a> Nature Reviews Cancer. 12 (8), 553-563 (2012).</li><li>Cheng, C. 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=YM155+as+an+inhibitor+of+cancer+stemness+simultaneously+inhibits+autophosphorylation+of+epidermal+growth+factor+receptor+and+G9a-mediated+stemness+in+lung+cancer+cells.">YM155 as an inhibitor of cancer stemness simultaneously inhibits autophosphorylation of epidermal growth factor receptor and G9a-mediated stemness in lung cancer cells.</a> PLoS One. 12 (8), 0182149 (2017).</li><li>Prasetyanti, P. R., Medema, 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=Intra-tumor+heterogeneity+from+a+cancer+stem+cell+perspective.">Intra-tumor heterogeneity from a cancer stem cell perspective.</a> Molecular Cancer. 16 (1), 41 (2017).</li><li>Zhao, Y., 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=CD133+expression+may+be+useful+as+a+prognostic+indicator+in+colorectal+cancer,+a+tool+for+optimizing+therapy+and+supportive+evidence+for+the+cancer+stem+cell+hypothesis:+a+meta-analysis.">CD133 expression may be useful as a prognostic indicator in colorectal cancer, a tool for optimizing therapy and supportive evidence for the cancer stem cell hypothesis: a meta-analysis.</a> Oncotarget. 7 (9), 10023-10036 (2016).</li><li>Choi, J. E., 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=Expression+of+epithelial-mesenchymal+transition+and+cancer+stem+cell+markers+in+colorectal+adenocarcinoma:+Clinicopathological+significance.">Expression of epithelial-mesenchymal transition and cancer stem cell markers in colorectal adenocarcinoma: Clinicopathological significance.</a> Oncology Reports. 38 (3), 1695-1705 (2017).</li><li>Massard, C., Deutsch, E., Soria, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+stem+cell-targeted+treatment:+elimination+or+differentiation.">Tumour stem cell-targeted treatment: elimination or differentiation.</a> Annals of Oncology. 17 (11), 1620-1624 (2006).</li><li>Grillet, 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=Circulating+tumour+cells+from+patients+with+colorectal+cancer+have+cancer+stem+cell+hallmarks+in+ex+vivo+culture.">Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture.</a> Gut. 66 (10), 1802-1810 (2017).</li><li>Dallas, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoresistant+colorectal+cancer+cells,+the+cancer+stem+cell+phenotype,+and+increased+sensitivity+to+insulin-like+growth+factor-I+receptor+inhibition.">Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition.</a> Cancer Research. 69 (5), 1951-1957 (2009).</li><li>Chang, Y. 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=STAT3+induces+G9a+to+exacerbate+HER3+expression+for+the+survival+of+epidermal+growth+factor+receptor-tyrosine+kinase+inhibitors+in+lung+cancers.">STAT3 induces G9a to exacerbate HER3 expression for the survival of epidermal growth factor receptor-tyrosine kinase inhibitors in lung cancers.</a> BMC Cancer. 19 (1), 959 (2019).</li><li>Catalano, 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=Colorectal+cancer+stem+cells+and+cell+death.">Colorectal cancer stem cells and cell death.</a> Cancers (Basel). 3 (2), 1929-1946 (2011).</li><li>Piggott, L., 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=Suppression+of+apoptosis+inhibitor+c-FLIP+selectively+eliminates+breast+cancer+stem+cell+activity+in+response+to+the+anti-cancer+agent,+TRAIL.">Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL.</a> Breast Cancer Research. 13 (5), 88 (2011).</li><li>Chung, S. Y., 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=Two+novel+SHP-1+agonists,+SC-43+and+SC-78,+are+more+potent+than+regorafenib+in+suppressing+the+in+vitro+stemness+of+human+colorectal+cancer+cells.">Two novel SHP-1 agonists, SC-43 and SC-78, are more potent than regorafenib in suppressing the in vitro stemness of human colorectal cancer cells.</a> Cell Death Discovery. 4, 25 (2018).</li><li>Cheng, C. 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=STAT3+exacerbates+survival+of+cancer+stem-like+tumorspheres+in+EGFR-positive+colorectal+cancers:+RNAseq+analysis+and+therapeutic+screening.">STAT3 exacerbates survival of cancer stem-like tumorspheres in EGFR-positive colorectal cancers: RNAseq analysis and therapeutic screening.</a> Journal of Biomedical Science. 25 (1), 60 (2018).</li><li>Kleist, B., Xu, L., Li, G., Kersten, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+adult+intestinal+stem+cell+marker+Lgr5+in+the+metastatic+cascade+of+colorectal+cancer.">Expression of the adult intestinal stem cell marker Lgr5 in the metastatic cascade of colorectal cancer.</a> International Journal of Clinical and Experimental Pathology. 4 (4), 327-335 (2011).</li><li>Medema, 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=Targeting+the+Colorectal+Cancer+Stem+Cell.">Targeting the Colorectal Cancer Stem Cell.</a> New England Journal of Medicine. 377 (9), 888-890 (2017).</li><li>Sahlberg, S. H., Spiegelberg, D., Glimelius, B., Stenerlow, B., Nestor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cancer+stem+cell+markers+CD133,+CD44,+CD24:+association+with+AKT+isoforms+and+radiation+resistance+in+colon+cancer+cells.">Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.</a> PLoS One. 9 (4), 94621 (2014).</li><li>Xia, J., Gill, E. E., Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NetworkAnalyst+for+statistical,+visual+and+network-based+meta-analysis+of+gene+expression+data.">NetworkAnalyst for statistical, visual and network-based meta-analysis of gene expression data.</a> Nature Protocols. 10 (6), 823-844 (2015).</li><li>Gagan, J., Van Allen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+to+guide+cancer+therapy.">Next-generation sequencing to guide cancer therapy.</a> Genome Medicine. 7 (1), 80 (2015).</li><li>Panichnantakul, P., Bourgey, M., Montpetit, A., Bourque, G., Riazalhosseini, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+as+a+Tool+to+Study+the+Tumor+Microenvironment.">RNA-Seq as a Tool to Study the Tumor Microenvironment.</a> Methods in Molecular Biology. 1458, 311-337 (2016).</li><li>Kanehisa, M., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KEGG+Mapper+for+inferring+cellular+functions+from+protein+sequences.">KEGG Mapper for inferring cellular functions from protein sequences.</a> Protein Science. 29 (1), 28-35 (2020).</li><li>Ashburner, 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=Gene+ontology:+tool+for+the+unification+of+biology.+The+Gene+Ontology+Consortium.">Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.</a> Nature Genetics. 25 (1), (2000).</li><li>The Gene Ontology Collective. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Gene+Ontology+Resource:+20+years+and+still+GOing+strong.">The Gene Ontology Resource: 20 years and still GOing strong.</a> Nucleic Acids Research. 47, 330-338 (2019).</li><li>Mi, H., Muruganujan, A., Thomas, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PANTHER+in+2013:+modeling+the+evolution+of+gene+function,+and+other+gene+attributes,+in+the+context+of+phylogenetic+trees.">PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees.</a> Nucleic Acids Research. 41, 377-386 (2013).</li><li>Yu, F., Shen, X. Y., Fan, L., Yu, Z. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+genetic+variations+assisted+by+Ingenuity+Pathway+Analysis+to+comprehensively+investigate+potential+genetic+targets+associated+with+the+progression+of+hepatocellular+carcinoma.">Genome-wide analysis of genetic variations assisted by Ingenuity Pathway Analysis to comprehensively investigate potential genetic targets associated with the progression of hepatocellular carcinoma.</a> European Review for Medical and Pharmacological Sciences. 18 (15), 2102-2108 (2014).</li><li>Zhou, 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=NetworkAnalyst+3.0:+a+visual+analytics+platform+for+comprehensive+gene+expression+profiling+and+meta-analysis.">NetworkAnalyst 3.0: a visual analytics platform for comprehensive gene expression profiling and meta-analysis.</a> Nucleic Acids Research. 47 (1), 234-241 (2019).</li><li>Cheng, C. 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=Epidermal+growth+factor+induces+STAT1+expression+to+exacerbate+the+IFNr-mediated+PD-L1+axis+in+epidermal+growth+factor+receptor-positive+cancers.">Epidermal growth factor induces STAT1 expression to exacerbate the IFNr-mediated PD-L1 axis in epidermal growth factor receptor-positive cancers.</a> Molecular Carcinogenesis. 57 (11), 1588-1598 (2018).</li><li>Gallego Romero, I., Pai, A. A., Tung, J., Gilad, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq:+impact+of+RNA+degradation+on+transcript+quantification.">RNA-seq: impact of RNA degradation on transcript quantification.</a> BMC Biology. 12, 42 (2014).</li></ol>

In this study, cultured cancer stem-like tumorspheres were used as a model in analyzing RNAseq data with available bioinformatics. For a disease model, HT29-derived tumorspheres were used. Because the tumorspheres have drug resistance against tumor therapies, the established model can be used to investigate the detailed mechanisms of resistance by investigating differences in gene expression. Moreover, genomic technology using RNAseq with available bioinformatics provides rapid understanding of the study model so the gen...

Colorectal cancer (CRC) is a leading cause of death with high prevalence and mortality worldwide1,2. Due to gene mutations and amplifications, cancer cells grow without proliferative control, which contributes to cell survival3, anti-apoptosis4, and cancer stemness5,6,7. Within a tumor tissue, tumor heterogeneity allows tumor cells to adapt and survive during therapeutic treatments8. Cancer stem cells (CSCs), with a higher rate of self-renewal and pluripotency than differential cancer types, are principally responsible for tumor recurrence9,10 and metastatic CRC11. CSCs present more drug resistance12,13,14 and anti-apoptosis properties15,16, thus surviving tumor chemotherapies.
Here, in order to investigate the potential mechanism for stemness in the selected CRC stem cells, RNAseq was performed to screen differentially expressed genes in tumor spheroids. The cancer cells can form spheroids (also called tumorspheres) when grown in low adherence conditions and stimulated by growth factors added to the cultured medium, including EGF, bFGF, HGF, and IL6. Therefore, we selected CRC HT29 tumor cells that resist chemotherapies with an increase in phosphorylated STAT3 when treated with oxaliplatin and irinotecon17. In addition, HT29 expressed higher stemness markers when cultured in the described culture conditions. The HT29-derived CSC model expressed higher amounts of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)18, a specific marker of CRC stem cells19,20. Moreover, CD133, considered a general biomarker for cancer stem cells, is also highly expressed in the HT29 cell line21. This protocol&#39;s purpose is to discover groups of driver genes in the established cancer stem-like tumorspheres based on bioinformatics datasets as opposed to investigating individual oncogenes22. It investigates potential molecular mechanisms through RNAseq analysis followed by available bioinformatics analyses.
Next generation sequencing is a high-throughput, easily available, and reliable DNA sequencing method based on computational help, used to comprehensively screen driver genes for guiding tumor therapies23. The technology is also used for detecting gene expression from reverse transcription of an isolated RNA sample24. However, when screening with RNAseq, the most important genes to target with therapy may not have the highest expression differential between experimental and control samples. Therefore, some bioinformatics were developed for classifying and identifying genes based on current datasets such as KEGG25, GO26,27, or PANTHER28, including Ingenuity Pathway Analysis (IPA)29 and NetworkAnalyst30. This protocol shows the integration of RNAseq and NetworkAnalyst to quickly discover a group of genes in the selected HT29-derived spheroids compared to parental HT29 cells. Application of this method to other disease models is also suggested for discovering differences in important genes.
Compared to investigation of individual gene expression, a high-throughput technique provides advantages to find potential driver genes easily for tumor precision medicine. With useful datasets such as KEGG, GO, or PANTHER, specific genes can be identified based on the disease models, signaling pathways, or specific functions, and this allows quickly focusing on specific, important genes, saving time and research costs. A similar application is used in previous studies14,18,31. Particularly, a tumor is more complicated because different types of tumors express distinguishing genes and pathways for survival and proliferation. Therefore, this protocol can pick up genes distinguishing different tumor types under different circumstances. There is the potential to find effective strategies against cancers by understanding the mechanism of specific gene expression.

<table>
	<tbody>
		<tr>
			<td>iRiS Digital Cell Imaging System</td>
			<td>Logos Biosystems, Inc</td>
			<td>I10999</td>
			<td>for observing the formation of tumorspheres</td>
		</tr>
		<tr>
			<td>Flow cytometry</td>
			<td>BD biosciences</td>
			<td>FACSCalibur</td>
			<td>for detecting the LGR5 and CD133 in the tumorspheres</td>
		</tr>
		<tr>
			<td>anti-LGR5-PE</td>
			<td>Biolegend</td>
			<td>373803</td>
			<td>LGR5 detection reagent</td>
		</tr>
		<tr>
			<td>anti-CD133-PE</td>
			<td>Biolegend</td>
			<td>372803</td>
			<td>CD133 detection reagent</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>GenScript</td>
			<td>Z00333</td>
			<td>for culture of tumorspheres</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>GenScript</td>
			<td>Z03116</td>
			<td>for culture of tumorspheres</td>
		</tr>
		<tr>
			<td>HGF</td>
			<td>GenScript</td>
			<td>Z03229</td>
			<td>for culture of tumorspheres</td>
		</tr>
		<tr>
			<td>IL6</td>
			<td>GenScript</td>
			<td>Z03034</td>
			<td>for culture of tumorspheres</td>
		</tr>
		<tr>
			<td>PureLink RNA extraction kit</td>
			<td>Invitrogen</td>
			<td>12183025</td>
			<td>isolate total RNA for RNAseq analysis</td>
		</tr>
		<tr>
			<td>RNAseq performance</td>
			<td>Biotools, Taiwan</td>
			<td></td>
			<td>RNAseq analysis is done commerially by Biotools, Ttaiwan</td>
		</tr>
		<tr>
			<td>NetworkAnalyst</td>
			<td>Institute of Parasitology, McGill University, Montreal, Quebec, Canada</td>
			<td></td>
			<td>http://www.networkanalyst.ca/</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>a statistical analysis software</td>
		</tr>
	</tbody>
</table>

discovery of driver genes in colorectal ht29-derived cancer stem-like tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

To establish the model for investigating the mechanism in cancer stem cells, colorectal HT29 cells were used to culture the cancer stem-like tumorspheres in vitro in a low-attachment plate containing B27, EGF, bFGF, HGF, and IL6. The tumorspheres &#62;100 &#181;m in diameter were formed in 7 days (Figure 1A). The tumorspheres were trypsinized to single cells and analyzed using flow cytometry to detect LGR5 and CD133 expression. LGR5 increased in the HT29-drived tumorspheres from 1.1% to 11.4...

Watch this Scientific Journal Video about Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres at JoVE.com

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Our protocol utilized RNA-Seq and bioinformatical tools to quickly uncover the overexpressed driver genes that contribute to the formation of stem-like colorectal tumorspheres. This protocol is used to screen a differentially expressed gene and signal pathways to investigate a molecular mechanism for cancer stemness in a selective colorectal cancers. On day seven of culture, use an inverted microscope with a digital cell imaging system to observe and measure the diameter of the tumorspheres.When the tumorspheres reach a greater than 100 micrometer diameter, treat the culture with 0.25%trypsin. After five minutes at 37 degrees Celsius, neutralize the trypsin with two times the volume of growth medium and count the cells using a hemocytometer. Collect the cells by centrifugation.And resuspend the pellet at a five times 10 to the four cells per 100 microliters of medium concentration. Add two microliters of the primary antibody of interest to each tube and incubate the cells for 30 minutes at room temperature and 200 revolutions per minute. At the end of the incubation, wash each tube with 900 microliters of PBS and analyze the cells for expression of the protein of interest by flow cytometry according to standard protocols.For RNA isolation after trypsin collection as just demonstrated, resuspend the cells at a two times 10 to the fifth cells per 50 microliters of PBS concentration and lyse each sample with 200 microliters of lysis buffer supplemented with two microliters of beta mercaptoethanol per tube. After brief vortexing and a five minute incubation at room temperature, collect the lysates by centrifugation and add 200 microliters of 70%ethanol to each tube. Transfer the samples into rapid columns and remove the solvent by centrifugation.Wash the samples with 400 microliters of wash solution one and 600 microliters of wash solution two to completely remove any non-RNA by centrifugation, followed by an additional two minutes centrifugation under the same centrifuge conditions to remove any residual ethanol. Then resuspend the pellets in 50 microliters of distilled water per tube and centrifuge the sample for one minute before collecting the RNA containing supernatants. To investigate the differential gene expression between tumorsphere cells compared to parental tumor cells after RNA sequencing analysis.Select genes with a greater than one to minus one log2 fold change with read counts greater than 100 in the tumorsphere and parental groups. And use the commands as indicated. To obtain a volcano plot of the data to determine the differential gene expression.For drive gene selection in network analyst, select single gene input and copy and paste the selected overexpressed genes with human specified as the organism and official gene symbol as the ID type. Click Upload and proceed. To insert and analyze the data using protein-protein interaction, following genetic protein-protein interaction, use the STRING interactome database with a confidence score cut off of 900 to show the seed genes.Cross-linking the uploaded genes. The seed genes associating with more individual genes can be selected as driver genes that may be involved in maintaining formation of the tumorsphere. Select Proceed in the mapping overview white in the Background and Force Atlas in the Layout knob.Then select PANTHER base pair to analyze the upregulation gene group. Tumorspheres of greater than 100 micrometers in diameter form within seven days of culture. Flow cytometric analysis reveals an increase in LGR5 and CD133 expression in HT29-derived tumorspheres compared to parental HT29 cells.After RNA sequencing, the genes with a greater than one log2 fold change in their up or downregulation with a P-value less than 0.05 as determined by heat map can be plotted to distinguish the significant genes between HT29-derived tumorspheres and parental HT29 cells. In this representative experiment, analysis of the upregulated genes with a greater than one log2 fold resulted in 10 seed genes, cross-linking the gene networks. PANTHER base pair analysis indicated that two genes were associated with negative regulation of apoptosis.Moreover, the selected genes were consequently validated by quantitative PCR confirming their increased expression in HT29-derived tumorspheres. The differential gene selection is the most important step of the procedures. Remember to use a log2 fold change greater than one with recounts greater than 100 as the criteria.Using this protocol, the genes selected from the colorectal-derived tumorspheres can be knocked down or overexpressed to uncover their potential roles in tumorsphere formation.

discovery-driver-genes-colorectal-ht29-derived-cancer-stem-like

Research

JoVE Journal

Cancer Research

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

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 ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...