Name: cell-free dna integrity analysis in urine samples
Uploaded: 2017-01-05T14:00:00
Description: Watch this Scientific Journal Video about Cell-Free DNA Integrity Analysis in Urine Samples at JoVE.com

The authors thank Gr&#225;inne Tierney and Silvia Bellissimo for their editorial assistance.

The protocol follows the guidelines of the IRST Human Research Ethics Committee.
NOTE: In this protocol, we isolated DNA from urine samples to perform a UCF DNA integrity analysis. Lncap and MRC cell lines were used to construct standards. Techniques such as DNA extraction, DNA quantification (spectrophotometer and real-time PCR for the control gene, STOX1), and real-time PCR for specific oncogenes were performed (Figure 1).
1. Urine Collection and Pr...

<ol>
	<li>Francis, G., Stein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Cell-Free+Tumour+DNA+in+the+Management+of+Cancer.">Circulating Cell-Free Tumour DNA in the Management of Cancer.</a> Int.J.Mol.Sci. 16 (6), 14122-14142 (2015).</li><li>Bryzgunova, O. E., Laktionov, P. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Her-2/neu+oncogene+amplification+in+clinically+localised+prostate+cancer.">Her-2/neu oncogene amplification in clinically localised prostate cancer.</a> J.Clin.Patho. 55 (2), 118-120 (2002).</li><li>Nord, H., 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=Focal+amplifications+are+associated+with+high+grade+and+recurrences+in+stage+Ta+bladder+carcinoma.">Focal amplifications are associated with high grade and recurrences in stage Ta bladder carcinoma.</a> Int.J.Cancer. 126 (6), 1390-1402 (2010).</li><li>Tabach, 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=Amplification+of+the+20q+chromosomal+arm+occurs+early+in+tumorigenic+transformation+and+may+initiate+cancer.">Amplification of the 20q chromosomal arm occurs early in tumorigenic transformation and may initiate cancer.</a> PLoS One. 6 (1), e14632 (2011).</li></ol>

UCF DNA integrity analysis is a new, non-invasive method for assessing DNA integrity in urine. It was recently proposed for the early diagnosis of bladder9 and prostate cancers7,8. A number of advantages and disadvantages of the UCF DNA integrity test are discussed here, together with future prospects.
The main advantage of the approach is that it offers an inexpensive, non-invasive method and a simple protocol to study urine as a potential source of biomarkers, requiring...

Cell-free DNA can be detected in blood and urine due to cell death by apoptotic or necrotic mechanisms. Cell-free DNA in blood has been widely studied for diagnostic and prognostic purposes in various diseases, especially cancer1. However, less is known about the role of urinary cell-free (UCF) DNA. UCF DNA may originate from blood passing through the glomerular filtration system or from cells that come directly into contact with this body fluid 2 (e.g., urothelial cells or prostatic cells). The use of UCF DNA as a source of biomarkers has mainly been investigated for the early diagnosis of renal, bladder, and prostate cancer due to the high percentage of UCF DNA coming directly from urinary tract cells3,4.
Little is known about UCF DNA and the best methods for isolating and characterizing it. Given the hypothesis that tumor cells release longer DNA fragments than normal cells, the evaluation of cell-free DNA integrity has been studied in an attempt to elucidate the origin of DNA in the blood circulation5. Some studies have demonstrated that cell-free DNA integrity in blood represents a good diagnostic test for many types of cancer6, and the same hypothesis has been proposed in relation to urine7-9.
This paper describes a new method for UCF DNA integrity analysis with a potential application to bladder and prostate cancer detection. In particular, the integrity of UCF DNA fragments longer than 250 bp was tested in 4 regions known to have an increased DNA copy number in solid tumors, including prostate and bladder cancer: c-MYC (8q24.21), HER2 (17q12.1), BCAS1 (20q13.2), and AR (Xq12)10-14. Specific oncogenes, rather than random sequences, were chosen to increase the probability of finding them in the cell-free fraction of cancer patients. One of the main advantages of this method is that it is flexible and that other regions can also be selected on the basis of tumor type and characteristics.

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">QIAamp DNA Mini Kit 
			&#160;</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">51304</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">iQ SYBR&#160;Green Supermix, 100 x 50 &#181;l rxns, 2.5 ml (2 x 1.25 ml)</td>
			<td style="width:216px;">Biorad</td>
			<td style="width:119px;">1708880</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">IDT custom DNA oligos&#160;</td>
			<td style="width:216px;">IDT&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">HPLC purification, 100nMole DNA oligo</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">NanoDrop 1000 Spectrophotometer</td>
			<td style="width:216px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Other spectrophotometric methods could also be used to quantify DNA</td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:277px;">Rotor-Gene 6000</td>
			<td style="width:216px;">Corbett</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Another Real Time PCR instrument could also be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">microcentrifuge</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">one centrifuge for 50 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">incubator</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">&#160;-80&#176;C freezer</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">-20&#176;C freezer</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">10 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">20 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">200 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">1000 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">pipette tips (10;20;200;1000)</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">1,5 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">50ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">15 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Rotor-Disc 72 Rotor</td>
			<td style="width:216px;">Corbett</td>
			<td>&#160;9018899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Strip Tubes and Caps, 0.1 ml (250)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">981103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Collection Tubes (2 ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Buffer AL (264 ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Proteinase K (10ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19133</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-free dna integrity analysis in urine samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

The total free DNA concentration was quantifiable by spectrophotometry for all samples analyzed, showing a range of between 1.51 and 138 ng/&#181;L. Five control samples were used for reproducibility of the data: two independent real-time experiments were performed for c-MYC, HER2, BCAS1, AR, and STOX1. The coefficients of variation (CV) were then calculated for each gene (Table 2), with a good degree of reproducibility between...

Watch this Scientific Journal Video about Cell-Free DNA Integrity Analysis in Urine Samples at JoVE.com

Cell-Free DNA Integrity Analysis in Urine Samples

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

The overall goal of this method is to assess the integrity of urine cell-free DNA at specific oncogene sequences. This method can help answer key questions in the urological cancer field, such as early non-invasive diagnosis of bladder and prostate cancer. The main advantage of this technique is that it is easy to do and to analyze.The implications of this technique extend towards diagnosis of urological malignancies, because they normally release cells and free nucleic acids in urine. Though this method can provide insight into urinary samples, it can also be applied to other systems such as serum or plasma. Generally, individuals new to this method would struggle because the concentration of urine cell-free DNA is often low.We first had the idea for this method when we tried to find a simple method for non-invasive early detection of bladder cancer. To begin the procedure, obtain a 50 milliliter clean catch first morning urine sample that has been stored at four degrees Celsius for no more than three hours. Invert the sample twice and then partition the sample into two 50 milliliter conical centrifuge tubes.Centrifuge the sample at 850 times G for 10 minutes at four degrees Celsius. Then, for each tube transfer 10 milliliters of the upper part of the supernatant to two five milliliter tubes, leaving at least two milliliters of supernatant above the pellet. Discard the pellets and store the supernatant at minus 80 degrees Celsius.Repeat this process for additional urine samples as needed. To begin DNA purification, for each sample thaw an aliquot of urine supernatant. Vortex the thawed sample and invert the tube twice.Place one milliliter of the sample into a five milliliter tube, and store the remainder at minus 80 degrees Celsius. Prepare a DNA purification kit suitable for urine samples according to the manufacturer's instructions. Add 100 microliters of proteinase K and one milliliter of lysis buffer to the sample and mix well with a pipette.Incubate the sample at 56 degrees Celsius for 15 minutes. During incubation, prepare the wash buffers in a spin column. After incubation, allow the sample to cool to room temperature and then add one milliliter of absolute ethanol and mix well by pipetting.Transfer 650 microliters of the sample to the column. Centrifuge at 6000 times G for one minute. Replace the collection tube and repeat this process until all of the sample has been loaded onto the column.Then add 500 microliters of the first wash buffer and centrifuge the column at 6000 times G for one minute. Replace the collection tube, add 500 microliters of the second wash buffer, and centrifuge the column at max speed for three minutes. Place the column in a clean collection tube and centrifuge at max speed for another three minutes to remove residual wash buffer.Transfer the column to a clean 1.5 milliliter micro centrifuge tube. Load 50 microliters of elution buffer onto the column and allow the buffer to soak into the column for seven minutes. Then centrifuge the column at 6000 times G for one minute.Pipette the eluate back into the column and centrifuge the column at max speed for one minute to obtain the urine cell-free DNA. To begin preparing the standards, first isolate DNA from an appropriate cell line using a purification kit. Quantify the DNA from each purified urine sample in the cell line with a spectrophotometer per the kit manufacturer's instructions.Dilute the UCF DNA to one nanogram per microliter and store the diluted samples at minus 20 degrees Celsius until ready for the integrity test. Dilute the cell line DNA to create six, 100 microliter standards of different concentrations. Store the standards at minus 20 degrees Celsius.To begin the DNA integrity test, thaw the UCF DNA samples, the standards, the appropriate PCR primers, and green supermix on ice. Then set up the 72-well rotor disc plate with strip tubes. Place two, 10 microliter aliquots of standard and then each sample into wells.Prepare two wells of 10 microliter RNase-free water as negative controls. For each filled well, prepare a mix of one microliter of each primer, 12.5 microliters of green supermix, and 6.5 microliters of RNase-free water. Transfer 15 microliters of the mix into each filled well, and then transfer the wells into a 72-well rotor disc.Perform the PCR with a realtime instrument according to the manufacturer's specifications. Using the instrument software, check if any replicates have a cycling threshold value difference of one or more. Discard those samples from the analysis and evaluate the median cycling threshold for the remaining samples.Perform melting analysis on each sample with a median cycling threshold value of 36 or less to evaluate the PCR product specificity. Then use the standard curve to determine a concentration in nanograms per microliter for each applicon. Urine cell-free DNA was isolated and the DNA integrity was evaluated in four regions that show increased DNA copy number in solid tumors.A control sequence was also evaluated to verify that the DNA quantity in each sample was sufficient for the integrity test. The total free DNA concentration and the coefficients of variation were calculated for each gene. This method shows 73%sensitivity and 84%specificity for detecting bladder cancer, but only 58%sensitivity and 44%specificity for prostate cancer.Once mastered, this technique can be done in one working day if it is performed properly. Following this procedure, other methods like digital PCR or next generation sequencing can be performed in order to answer additional questions like copy number inducer of the genes of interest. After watching this video you should have a good understanding of how to isolate cell free DNA from urine in a very simple manner, and how to study the integrity of specific regions of interest using real time PCR approaches.

cell-free-dna-integrity-analysis-in-urine-samples

Research

JoVE Journal

Cancer Research

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 ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

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 ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

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 ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

In Vivo Brain Tumor Modeling via Electroporation-Based Delivery of Plasmid DNA

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

Author Spotlight: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures  

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in ...