The authors would like to acknowledge the generosity of all patients and their families. This work was supported by funding from the Smashing Walnuts Foundation (Middleburg, VA), the V Foundation (Atlanta, GA), The Gabriella Miller Kids First Data Resource Center, Clinical and Translational Science Institute at Children&#39;s National (5UL1TR001876-03), Musella Foundation (Hewlett, NY), Matthew Larson Foundation (Franklin Lake, NJ), The Lilabean Foundation for Pediatric Brain Cancer Research (Silver Spring, MD), the Childhood Brain Tumor Foundation (Germantown, MD), the Children&#39;s Brain Tumor Tissue Consortium (Philadelphia, PA), and Rally Foundation for Childhood Cancer Research (Atlanta, GA).

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The method described in this manuscript is included in an International patent application "Methods for detecting cancer biomarkers" by Javad Nazarian &amp; Eshini Panditharatna.

Here we have presented our method for detecting and quantifying the allelic frequency of tumor mutations in cfDNA from&#160;patient liquid biopsy. We emphasize critical steps for the success of this method, including pre-analytical sample processing, cfDNA extraction, PCR assay design, and data analysis. To limit the sample volume used, cfDNA is extracted from 1 mL of plasma but only 500 &#181;L of CSF. When extracting from CSF, the protocol for extraction from 1 mL of urine (following the cfDNA extraction kit handbook12) is used, as per the manufacturer&#39;s recommendation. The difference in sample volume required between plasma and CSF is due to lower levels of tumor-specific cfDNA in plasma compared to CSF of patients with brain tumors, necessitating larger sample volumes for mutation detection8. If the sample is available, more than 1 mL of plasma may be extracted from to produce higher DNA yield. However, in the case of pediatric patients, it is important to minimize the amount of blood used whenever possible, as even a simple procedure such as blood draw is fatiguing to pediatric patients with cancers undergoing radiation therapies. Extracting from 1 mL aliquots of plasma also enables replicate extractions, such that the assay can be repeated (e.g., in cases of failure in DNA extraction or sample contamination).
In an effort to reduce any sample use that is not strictly necessary, cfDNA is typically not quantified. However, we have found a range of cfDNA concentrations between 0.2-2 ng/&#181;L and 0.6-13 ng/&#181;L in plasma and CSF, respectively. Given the low amount of cfDNA, and the fact that tumor-specific mutant alleles are present at a low frequency, a pre-amplification step using the same set of primers used for dPCR is necessary to significantly increase the amount of target DNA in the sample, aiding in mutation detection8. Diluting the pre-amplified product in DNA suspension buffer provides sufficient volume for technical replicates, which aids in distinguishing true positives. Because the number of mutant droplets can be low (for example, between 0-2 mutant droplets in a plasma sample), the inclusion of technical replicates is key for resolving mutation status. While one PCR well may yield 0 mutant droplets, the other two may yield 1-2 for a single plasma sample analyzed in triplicate; thus, the inclusion of replicates allows for greater accuracy when determining mutation status. The MAF is then calculated as the average of the replicate values.
Multiplexed pre-amplification (preamplifying wildtype and mutant alleles of two mutations of interest) increases the utility of a single sample, as the same starting material can be used to test for two mutations. Importantly, a multiplex preamplification product can be analyzed in singleplex during dPCR for greater simplicity, as described here. However, both preamplification PCR and dPCR may be multiplexed. When multiplexing, conditions must be optimized for both sets of primers and probes: primer annealing temperatures must be similar to run together in PCR amplification, and probes should be designed to generate distinct clusters (based on fluorescent signal and intensity). When validating a new set of primers and probes, run an annealing temperature gradient to determine optimum annealing temperature based on the range suggested by the manufacturer. Before testing probes on patient cfDNA specimens, validate them using synthetic DNA constructs and/or tumor tissue gDNA of different inputs (i.e., up to 10 ng).
For the detection of mutant alleles with greater specificity and reduced mismatches in the hybridization of probe to the target DNA, locked nucleic acid (LNA) probes are used. LNA is a nucleic acid analog with a methylene bridge connecting the 2&#39;-oxygen and 4&#39;-carbon of the ribose ring9. The methylene bridge locks the nucleic acid into a rigid bicyclic conformation that restricts flexibility, increases thermal duplex stability, and improves the specificity of probe hybridization to target DNA10,18,19. If a single base mismatch exists between LNA probe and template DNA, duplex formation between probe and target will destabilize. As such, LNA probes improve the specificity of probe binding and result in a higher signal-to-noise ratio11. Analysis of plasma and CSF from non-CNS-diseased pediatric patients has established the specificity of our assay with LNA probes targeting H3F3A p.K27M, to determine that an allelic frequency of equal to or less than 0.001% is considered a false positive8. For additional considerations for optimizing the design of probes and primers, including GC content, amplicon size, probe reporter dyes, and quenchers, refer to the dPCR manufacturer guidelines14,17. Although our method is optimized for use with the RainDance system, the protocol may be adapted for use with other dPCR platforms.
The method presented here draws strength from the high sensitivity and target enrichment achieved by dPCR, which remains the platform of choice for detecting rare tumor mutations in cfDNA. Although powerful, dPCR is limited in the number of mutations that can be tested for in a single assay. An alternative to dPCR is next generation sequencing (NGS), which may detect multiple mutations across many genes, increasing the utility of a single sample. NGS can detect mutations in CSF and plasma of patients with brainstem gliomas20, however, is currently less sensitive than dPCR at detecting tumor mutations in cfDNA, with detection limits of 0.1-10% MAF compared to 0.001% in PCR-based approaches21. dPCR also achieves faster turnaround time than NGS, enabling rapid detection of mutations of interest.The PCR approach is applicable across cancer types and can be expanded to detect hotspot mutations and methylated cfDNA22.
The liquid biopsy is indeed in its infancy and will require further development for tailoring to specific diseases. Tumor monitoring in the context of tumor evolution will be the next challenge, where a platform capable of detecting emerging mutations with high sensitivity would be required. Additionally, platforms capable of detecting a variety of biomolecules (peptides, cytokines, RNA) will be highly beneficial in assessing tumor response to treatment, as well as advancing personalized clinical interventions.

Due to&#160;limitations in the availability of&#160;tumor tissue for molecular analyses, there is a need for the development of highly sensitive methods capable of detecting tumor somatic mutations in patient biofluids, including plasma, serum and cerebrospinal fluid (CSF). For example, pediatric diffuse midline gliomas (DMGs) present a challenge due to their neuroanatomical location. Over the past decade, molecular profiling of DMG tissue specimens has uncovered driver mutations in this tumor type1 and has revealed spatial and temporal heterogeneity of mutations, making biopsy necessary for characterizing a patient within a disease subgroup and promoting molecularly-targeted therapies2. As such, clinical trials advocate for integrating surgical biopsies for tumor molecular profiling at upfront diagnosis3,4,5,6. During&#160;treatment, monitoring of&#160;therapeutic response in DMGs is limited to MRI, which lacks sensitivity to detect small changes or tumor genomic evolution. MRI is also prone to detect pseudoprogression, transient inflammation at the site of the tumor that mimics true progression on imaging and may misinform interpretation of tumor response7. Thus, DMGs represent a tumor type with a high need for an alternative, minimally invasive means of detecting tumor mutations and monitoring clinical response. In order to address these needs, we developed and optimized a protocol for detecting, and quantifying the allelic frequency of, tumor mutations in cfDNA from plasma, serum and CSF of children with midline gliomas8. Given that childhood DMGs often (&#62;80%) harbor a somatic lysine-to-methionine mutation at position 27 of histone variant H3.1 (H3.1K27M, 20% of cases) or H3.3 (H3.3K27M, 60% of cases)1, these and other mutations characteristic of DMGs (i.e., ACVR1, PIK3R1) were targeted for mutant allele quantification using cfDNA8. This assay can be tailored to detect hotspot mutations in other tumor types using sequence-specific primers and probes. The versatility of this approach makes it applicable to a variety of cancers which would benefit from the integration of cfDNA-based diagnostic tools and therapeutic monitoring.
To sensitively detect low allelic frequency mutations in cfDNA, we employ a droplet digital PCR (dPCR)-based approach. In this method, the PCR reaction mixture is partitioned into a theoretical maximum of 10 million droplets using the dPCR platform (e.g., RainDance), with 7-9 million droplets per PCR well typically seen experimentally. Due to the high degree of&#160;sample partitioning, at most only one to two DNA molecules can be present in each droplet. PCR amplification and sequence-specific fluorescent probe hybridization can occur in each droplet, such that millions of reactions take place. This maximizes the sensitivity and enables detection of rare mutant alleles, which might otherwise go undetected against a background of wildtype DNA. The utilization of locked nucleic acid (LNA) probes enhances the specificity of the assay by restricting mismatch hybridization and supporting accurate mutation detection9,10,11. Here, we describe our methods of sample processing, cfDNA extraction, preamplification of target alleles, dPCR and data analysis.

<table border="0" cellpadding="0" cellspacing="0" style="width:1483px;" width="1482">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:458px;">10mM Tris-HCl, 0.1mM EDTA DNA Suspension Buffer (pH 8.0)</td>
			<td style="width:240px;">Teknova</td>
			<td style="width:123px;">T0227</td>
			<td style="width:663px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:458px;">BD Vacutainer K2-EDTA 7.2mg Blood Collection Tubes (10mL)</td>
			<td style="width:240px;">Becton Dickinson Diagnostics</td>
			<td>367525</td>
			<td style="width:663px;">For plasma collection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:458px;">BD Vacutainer Plastic Serum tube with Red BD Hemogard Closure (10mL)</td>
			<td style="width:240px;">Becton Dickinson Diagnostics</td>
			<td>367895</td>
			<td style="width:663px;">For serum collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bleach</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buffer ATL</td>
			<td>Qiagen</td>
			<td>19076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell-Free DNA Blood Collection Tubes</td>
			<td>Streck</td>
			<td>218961</td>
			<td>cfDNA blood collection tubes (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CentriVap Concentrator</td>
			<td>Labconco</td>
			<td>7810010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward Primer</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td></td>
			<td>Custom design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MiniAmp Thermal Cycler</td>
			<td>Thermofisher Scientific</td>
			<td>A37834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:458px;">Molecular Biology Grade Absolute Ethanol (200 proof)</td>
			<td style="width:240px;">General Lab Supplier</td>
			<td style="width:123px;"></td>
			<td style="width:663px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mutant Probe</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td></td>
			<td>Custom design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PAXgene Blood ccfDNA tubes</td>
			<td>PreAnalytiX</td>
			<td>768115</td>
			<td>cfDNA blood collection tubes (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR 8 well strip tube caps</td>
			<td>VWR</td>
			<td>10011-786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR 8 well strip tubes</td>
			<td>Axygen</td>
			<td>PCR-0208-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:458px;">Pipette (p20, p200, p1000)</td>
			<td>General Lab Supplier</td>
			<td style="width:123px;"></td>
			<td style="width:663px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette filter tips (p20, p200, p1000)</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:458px;">Q5 Hot Start High-Fidelity 2X Master Mix</td>
			<td>New England Biolabs Inc</td>
			<td>M0494S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp Circulating Nucleic Acid HandBook&#160;</td>
			<td>Qiagen</td>
			<td></td>
			<td>cfDNA extraction kit handbook (2013 edition)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp Circulating Nucleic Acid Kit</td>
			<td>Qiagen</td>
			<td>55114</td>
			<td>cfDNA extraction kit (Protocol Step 4)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp MinElute Virus Spin Kit</td>
			<td>Qiagen</td>
			<td>57704</td>
			<td>Optional kit for DNA extraction from small (&#60; or =200&#181;L) sample volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qiavac 24 Plus</td>
			<td>Qiagen</td>
			<td>19413</td>
			<td>For use with QIAamp Circulating Nucleic Acids kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Raindance Consumable Kit</td>
			<td>Bio-Rad</td>
			<td>20-04411</td>
			<td>Contains droplet-generating instrument chips, quantification instrument chips, PCR strip caps including high-speed caps, and droplet stabilizing oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Raindance Sense Instrument</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Quantification instrument (used in Protocol Step 9)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Raindance Source Instrument</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Droplet-generating instrument (used in Protocol Step 9)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RainDrop Analyst II Software</td>
			<td>RainDance Technologies</td>
			<td></td>
			<td>Analyst software used for Data Analysis (Protocol Step 10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated Vapor Trap</td>
			<td>Savant</td>
			<td>RVT5105-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse Primer</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td></td>
			<td>Custom design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Smartblock 2mL</td>
			<td>Eppendorf</td>
			<td>05 412 506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:458px;">TaqMan Genotyping Master Mix</td>
			<td>Thermofisher Scientific</td>
			<td style="width:123px;">4371353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer C</td>
			<td>Eppendorf</td>
			<td>14 285 562PM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WT Probe</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td></td>
			<td>Custom design</td>
		</tr>
	</tbody>
</table>

detection and monitoring of tumor associated circulating dna in patient biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Figure 1&#160;shows representative results for successful detection of H3F3A p.K27M mutation in preamplified plasma (top left panel) and CSF (top right panel) cfDNA from two children with DMG. cfDNA samples were analyzed in replicate but only one representative graph is shown for each sample type. The dPCR plots show successful droplet generation, with 7-9 million droplets per PCR well (most of which are negative droplets that do not contain target DNA). A minimum of 7 million droplets per PCR well indicates a successful dropletization, while fewer than 7 million indicate failure of the assay, in which case the user should not proceed with data analysis. Figure 1 shows a clear separation between mutant and wildtype clusters along the x and y-axes, respectively. Robust wildtype clusters indicate that the cfDNA extraction was successful because template DNA is present. Mutant clusters show a MAF of 1.60% and 39.92% for the plasma and CSF samples, respectively, demonstrating positive detection of the mutation. For these patients, dPCR results are in accordance with tumor mutation status, as confirmed by genomic analysis of biopsy tumor tissue8. The negative control (bottom left panel) shows 0 mutant and 0 wildtype droplets, indicating that there was no contamination of the PCR reaction mixture. The positive control tumor tissue gDNA (bottom right panel) shows the mutation being detected at the expected allelic frequency for the particular tumor sample selected.&#160;
In Figure 2, representative results are shown for unsuccessful detection of the mutation of interest, H3F3A p.K27M, in plasma cfDNA from a child with DMG. Mutation status was confirmed by genomic analysis of tumor tissue8. Representative results from two replicate PCR wells are shown (top panels). The total number of droplets, including negative droplets, is between 7-9 million per PCR well, indicating successful dropletization. The wildtype clusters, plotted along the y-axis, show approximately 7-8,000 wildtype droplets per PCR well for the plasma cfDNA, indicating successful cfDNA extraction (as there is target wildtype DNA in the sample). However, 0 mutant droplets are detected in the plasma sample. The bottom left panel shows negative control (MB H2O) with no wildtype or mutant droplets; and the bottom right panel shows the positive control preamplified tumor tissue gDNA (0.025 ng) with positive mutation detection. As shown in this figure, the absence of mutation detection in plasma may not necessarily mean the patient is wildtype for the mutation of interest, as false negatives do occur8. In some cases, when the mutation is missed in the plasma collected at upfront biopsy, it is detected in the plasma obtained from the same patient at a later time point8.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59721/59721fig1v2.jpg" /> 
Figure 1: Successful detection of H3F3A p.K27M mutation in preamplified plasma and CSF cfDNA from children with midline gliomas. <a href="https://www.jove.com/files/ftp_upload/59721/59721fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59721/59721fig2.jpg" /> 
Figure 2: False negative results obtained from analysis of a preamplified plasma cfDNA sample from a patient known to harbor the mutation of interest, H3F3A p.K27M, in the tumor. <a href="https://www.jove.com/files/ftp_upload/59721/59721fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids at JoVE.com

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

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Cancer Research

8.5K Views.  Children's National Health System. Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Video: Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

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 ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

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 ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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 ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...