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 DMEM with 10% FBS and 1% P/S.</li>
	<li>Count the HT29 cells using a hemocytometer.</li>
	<li>Add 2,000 cells/well to a low attached 6 well plates with 2 mL of serum-free DMEM with 1% P/S and supplemented with 0.2% B27, 20 ng/mL of epidermal growth factor (EGF), 20 ng/mL of fibroblast growth factor (bFGF), 20 ng/mL of hepatocyte growth factor (HGF), and 20 ng/mL of interleukin 6 (IL6).</li>
	<li>Grow the cells at 37 &#176;C with 5% CO2 and 95% humidity under aseptic conditions.</li>
	<li>Add 0.5 mL of cancer stem cell medium every 2 days until the tumorspheres measure &#62;100 &#956;m in diameter for at least 7 days.</li>
	<li>Observe and measure the tumorsphere diameter using an inverted microscope with a digital cell imaging system.</li>
	<li>When the tumorspheres reach &#62;100 &#956;m in diameter, trypsinize the tumorspheres with 0.25% trypsin for 5 min at 37 &#176;C and neutralize the trypsin by adding 2x the volume of growth medium. Count the cells using a hemocytometer.</li>
	<li>Centrifuge at 1,200 rpm for 10 min and remove the supernatant.</li>
	<li>Incubate 5 x 104 cells with 2 &#181;L of anti-LGR5-PE and 2 &#181;L of anti-CD133-PE in 100 &#181;L of DMEM individually for 30 min at room temperature, shaking at 200 rpm. 
	NOTE: CD133 is a general cancer stem cell biomarker that is highly expressed in HT29 cells.</li>
	<li>Add 900 &#181;L of PBS and analyze the LGR5 and CD133 expression using flow cytometry. A change in fluorescence in the FL2-H channel indicates gene expression.</li>
</ol>
2. RNA isolation
NOTE: Use a commercial kit (see Table of Materials) with a rapid column for RNA isolation following the manufacturer&#8217;s instructions.
<ol>
	<li>Add 50 &#181;L of PBS to the harvested cells (2 x 105 cells) and resuspend them with pipetting.</li>
	<li>Add 200 &#181;L of lysis buffer containing 2 &#181;L of beta-mercaptoethanol (&#946;-ME). Vortex quickly and let stand for 5 min.</li>
	<li>Centrifuge the solution at 16,000 x g for 10 min. Collect the supernatant and mix with 200 &#181;L of 70% ethanol.</li>
	<li>Use the attached column to remove the solvent by centrifugation at 14,000 x g for 1 min.</li>
	<li>Wash using wash solution 1 and 2 to completely remove non-RNAs by centrifugation at 14,000 x g for 1 min.</li>
	<li>Centrifuge again at 14,000 x g for 2 min to remove residual ethanol.</li>
	<li>Add 50 &#181;L of distilled water, centrifuge at 14,000 x g for 1 min, and collect the solution.</li>
	<li>Measure the RNA concentration using OD260 with a spectrophotometer. 
	RNA concentration (&#181;g/mL) = (OD260) x (40 &#181;g RNA/mL) and OD260/OD280 &#62; 2. RNA samples should have an RNA integrity number&#160;(RIN) &#62; 7.</li>
</ol>
3. RNAseq profiling and bioinformatics analysis
NOTE: RNAseq analysis was performed commercially (see Table of Materials) to investigate the differential genes in the HT29-derived tumorspheres compared to parental HT29 cells.
<ol>
	<li>Use commercial services for RNAseq steps, including library construction, library quality control, and DNA sequencing.</li>
	<li>The data report should contain important information, including the read counts, log2 fold change, and p value. Select the differential genes according to the following parameters: genes with a &#62; 1 log2 fold change with read counts &#62;100 in the HT29 tumorsphere group, and genes &#60;-1 log2 fold change with read count &#62;100 in the HT29 parental group. In this case, a p value &#60; 0.05 was considered acceptable and the data were used (Table 1). 
	NOTE: Here, a gene count &#62;100 was used as the threshold to continue the study of a particular gene and validate its expression.</li>
	<li>Use statistical analysis software (see Table of Materials), to show a heatmap and identify overexpressed genes &#62;1 and downregulated genes &#60; 1 in log2 fold change.</li>
	<li>Use R software to draw the Volcano Plot with x: log2 fold change; y: -log10 (p value) to show the differential genes.
	<ol>
		<li>Install R library 
		install.packages(library(calibrate))&#160;</li>
		<li>Read the data in RStudio with the following program: 
		res &#60;-read.csv(&#34;/Users/xxx.csv&#34;, header=T) 
		head(res) 
		with(res, plot(log2FoldChange, -log10(pvalue), pch=19, main=&#34;HT29CSC vs HT29&#34;, xlim=c(-6,6), col=&#34;#C0C0C0&#34;)) 
		with(subset(res, pvalue&#60;.05 &#38; log2FoldChange&#62;1), points(log2FoldChange, -log10(pvalue), pch=19, col=&#34;red&#34;)) 
		with(subset(res, pvalue&#60;.05 &#38; log2FoldChange&#60;(-1)), points(log2FoldChange, -log10(pvalue), pch=19, col=&#34;blue&#34;)) 
		with(subset(res, pvalue&#62;.05), points(log2FoldChange, -log10(pvalue), pch=19, col=&#34;#444444&#34;)) 
		abline(h=1.3, lty=2) 
		abline(v=1, lty=2) 
		abline(v=(-1), lty=2)</li>
		<li>Execute the run to obtain the Volcano Plot.</li>
	</ol>
	</li>
</ol>
4. Driver gene selection
<ol>
	<li>Select Single gene Input in NetworkAnalyst.</li>
	<li>Copy and paste the selected overexpressed genes from Table 1 with &#8220;human&#8221; specified as the organism and ID type Official Gene Symbol. 
	NOTE: Alternatively, use Ensembl Gene ID for copy and paste.</li>
	<li>Insert the data by clicking Upload and Proceed to analyze it using protein-protein interaction (PPI) following genetic PPI.</li>
	<li>Use the STRING interactome database with a confidence score cutoff of 900 to show the seed genes cross-linking the uploaded genes. The seed genes associating with more individual genes were selected as driver genes that may be involved in maintaining formation of HT29-derived tumorspheres. 
	NOTE: There are three interactome datasets for use: IMEx, STRING, and Rolland. STRING contains higher confidence experimental evidence. With lower uploaded gene numbers, IMEx can be selected to predict and pick up the driver genes in the interactome networks.</li>
	<li>Select Proceed in the mapping overview.</li>
	<li>Select White in the Background and Force Atlas in the Layout knob.</li>
	<li>Select PANTHER BP to analyze the upregulation gene group. 
	NOTE: This shows that HSPA5 was responsible for anti-apoptosis in the HT29-derived tumorspheres in this study (Figure 3A). To narrow down the specific functional field, KEGG, GO, or PANTHER classification can be used alternatively to select the specific driver genes.</li>
</ol>

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The authors have no relevant financial disclosures.

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>&lt;strong&gt;http://www.networkanalyst.ca/&lt;/strong&gt;</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...

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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 ...

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

Research

JoVE Journal

Cancer Research

6.5K Views.  Chang Gung University, Chang Gung Memorial Hospital at Linkou. Ons protocol maakte gebruik van RNA-Seq en bio-informatische tools om snel de overuitexpressie driver genen die bijdragen aan de vorming van stam-achtige colorectale tumorsferen bloot te leggen. Dit protocol wordt gebruikt om een differentieel uitgedrukt gen en signaaltrajecten te screenen om een moleculair mechanisme voor kankerstamness in een selectieve colorectale kanker te onderzoeken. Gebruik op dag zeven van de cultuur een omgekeerde microscoop met een digitaal celbeeldvormingssysteem o...

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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

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 ...

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells (CSCs) and identifying suitable molecular targets relies on methods that allow their specific discrimination in heterogeneous samples such as cell lines and ex vivo tumor tissue. Flow cytometry/FACS is a powerful technology to multi-parametrically dissect biological samples at the single cell level and is to date the method of choice to recover live cells for downstream analyses. Surface markers such as CD44 and CD133 as well as detection of aldehyde dehydrogenase enzymatic activity have often been used to define and sort out CSCs from tumor samples by FACS. A complementary approach, depicted here in methodological detail, makes use of functional dye extrusion by ABC drug transporters, which identifies a distinct population of fluorescence-dim cells commonly referred to as side population (SP). SP&#160;cancer cells exhibit canonical stem cell characteristics and can be abrogated and functionally confirmed using agents that inhibit the dye-extruding drug transporter (most frequently ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338). Moreover, the SP&#160;assay is compatible with other flow cytometric evaluations such as staining of surface antigens, aldehyde dehydrogenase detection and dead cell discrimination (e.g., with 7-AAD or propidium iodide (PI)). Thus, we describe a valuable and broadly applicable method for CSC identification, isolation and sub-characterization mechanistically based on a functional, rather than a phenotypic parameter. Although originally performed with Hoechst 33342 as triggering dye, we here focus on the more recent Violet dye-based SP phenotype that is resolvable on any flow cytometer equipped with a violet laser source.

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Genetics

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicine

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.

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 ...

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

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