Name: advanced animal model of colorectal metastasis in liver: imaging techniques and properties of metastatic clones
Uploaded: 2016-11-30T12:30:00
Description: Watch this Scientific Journal Video about Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones at JoVE.com

We would like to thank Dr. Geoffrey L. Greene (University of Chicago) for the Luc2-tdTomato plasmid and the HCT116 cell line, Mr. Ani Solanki (Animal Resource Center) for the mice management, and Dr. Lara Leoni for the assistance with the DLIT. Quantifications of fluorescent and luminescent intensities were performed in the Integrated Small Animal Imaging Research Resource at the University of Chicago on an IVIS Spectrum (PerkinElmer, Hopkinton, MA). This work was supported by the Virginia and D.K. Ludwig Fund for Cancer Research, the Lung Cancer Research Foundation (LCRF), the Prostate Cancer Foundation (PCF), and the Cancer Center Support Grant (P30CA014599). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Chicago (Protocol # 72213-09) and performed under sterile conditions.
1. Preparations
<ol>
	<li>Make 500 mL of medium for the culture of HCT116 tumor cells: Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin.</li>
	<li>Autoclave the instruments to be used for the spleen injection model, including 3 - 4 surgical towels,...

<ol>
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The animal model presented in the current report is based on two major approaches. First, in order to ensure the ability to observe metastatic clones with different propensities to colonize and proliferate in the liver, a panel of highly heterogeneous monoclonal cell lines was established, rather than an established unfractionated cancer cell line12,13. The monoclonal approach to metastasis development is justified by recent genomic data14 and was successfully used previously to model the metastatic...

Patients with liver metastases from primary colorectal cancers (CRC) are characterized by a poor prognosis. The 5-year survival rate for primary nonmetastatic CRC (stages I - III) is estimated as 75 - 88%3,4, while patients with liver metastases (stage IV) have a 5-year survival rate of only 8 - 12%5,6. However, metastatic patients represent a heterogeneous group, presenting with different numbers of metastases and different recurrence times. Clinical observations indicate that the number of metastases (which may be proportional to colonizing ability or frequency of colonization) and the size of any single metastasis (proportional to the local growth rate) are independent prognostic factors1,7. In other words, the success of metastatic clones colonizing the liver depends on two major properties: their ability to grow and their ability to disseminate and survive in the liver microenvironment.
The design of successful clinical models with the capability of capturing and quantifying the properties of metastatic clones can drastically improve our understanding of liver metastasis biology and provide an effective tool for the design of potential therapeutic approaches. Models of experimental liver metastasis have been previously reported8,9, but neither of them provided the ability to quantitatively capture and describe properties of individual metastatic clones both in vivo and ex vivo.
Here, we present a new, advanced model of liver metastasis that includes the generation of tumor clones with different liver colonization efficiencies and growth properties. We employed a combination of dual-labeling of cancer cells with luciferase and tdTomato fluorescent protein with the generation of monoclonal cell lines that have intrinsic differences in metastatic capacity. In this experimental model, the data indicate that the development of liver metastases can be described in terms of colonization frequency and doubling time (Td), which is consistent with clinical observations. The quantitative nature of this model makes it easily adoptable for drug discovery and diagnostic purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td>124262</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>LivingImage 4.0 Software</td>
			<td>Caliper Life Sciences</td>
			<td>128165</td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>VAD-MGX Research Anesthetic Machine</td>
			<td>Vetamac</td>
			<td>VAD-MGX</td>
			<td>Inhalation anesthesia machine</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid (10,000 units penicillin;10,000 &#956;g streptomycin)</td>
			<td>Invitrogen</td>
			<td>15140163</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>24020117</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>Buprenex Injection (0.3mg/mL)</td>
			<td>Reckitt Benckiser Healthcare Ltd.</td>
			<td>12496-0757-5</td>
			<td>Buprenorphine hydrochloride</td>
		</tr>
		<tr>
			<td>Gemini Cautery System</td>
			<td>Braintree Scientific</td>
			<td>GEM 5917</td>
			<td>Hand-held cautery for splenectomy</td>
		</tr>
		<tr>
			<td>Micro Clip; Straight; 70 Grams Pressure; 1.5mm Clip Width; 10mm Jaw Length</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5426</td>
			<td>Hemoclip: Hemostasis instruments after spleen injection</td>
		</tr>
		<tr>
			<td>D-luciferin, potassium salt</td>
			<td>Goldbio Technology</td>
			<td>LUCK-1G</td>
			<td>Luciferin potassium salt</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985062</td>
			<td>Reduced Serum Medium</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>BIO-RAD</td>
			<td>1450102</td>
			<td>Automatic cell counter</td>
		</tr>
		<tr>
			<td>JMP10 software&#160;</td>
			<td>SAS Institute</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>BD FACSAria II cell sorter</td>
			<td>BD Biocsiences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
	</tbody>
</table>

advanced animal model of colorectal metastasis in liver: imaging techniques and properties of metastatic clones

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

The goal of this experiment was to establish a consistent and easily reproducible animal model with the potential for the serial quantification of the in vivo metastatic tumor burden and for the estimation of the colonizing frequency and growth kinetics of developing liver metastases. Figures 2-6, with legends, are provided from our previous publication under a Creative Commons CC-BY license10.
...

Watch this Scientific Journal Video about Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones at JoVE.com

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

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

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

The overall goal of this approach is to model liver colonization by metastatic tumor clones generated by colorectal cancers, and to use this model to improve diagnostics and therapy of liver metastases. Clinical observations indicate the heterogeneity of metastatic disease. Our model provides an ability to capture this heterogeneity to detect genes which are associated with oligometastatic and polymetastatic disease.And to use this knowledge for improvement of early detection and treatment of metastasis. The main advantage of our technique is that we use double-labeled monoclonal cell lines derived from patients with colorectal cancer, and can observe development of liver metastases both in vivo and ex vivo. The implications of this technique extend toward a therapy of metastatic disease.It gives us the ability to test any potential drug with quantitative estimation of numbers of metastatic lesions and growth kinetics of each lesion. After generating luciferase tdTomato labeled HCT116 cells and preparing media, reagents, and instruments, according to the text protocol, plate the transpected cells at the following densities per well, in triplicate, in 96-well plates. Following the incubation of the cells for five hours, quantify the tdTomato fluorescent intensities using an IVIS system by starting the image software on the desktop.Click the Initialize button to initialize the imaging time, medium binning, two for F/Stop, 535 for the excitation filter, and 580 for the emission filter. Place the 96-well plate on the imaging station, and click on Acquire to start imaging. After the image is acquired, in the Tool Palette, click ROI tools and select 12x8 from the slit icon.Create 12x8 slits to cover the area of the signal on the image of the 96-well plate and click the Measurements tab. Observe a window with the table of ROI measurements with units of photons per second, per steradian, per square centimeter. Once the cells reach 70 to 80 percent confluency, approximately one hour prior to spleen injection, add 0.05 percent tripsin and 0.53 millimolar EDTA to the cells to dissociate them.After incubating the cells for three to five minutes, use an equal volume of C-DMEM to neutralize the tripsin. Thoroughly pipette the cultures to avoid clumping, then use an automatic cell counter to count the cells. Freeze suspended cells in 1x DBPS at a concentration of 1.2 to 2 x 10 to the 6 cells per 100 microliters, pipetting thoroughly to avoid clumping.Then, keep the cells on ice until injection, occasionally re-suspending them in the tube. After administering analgesics and anesthetizing a six to eight week old female athymic nude mouse according to the text protocol, identify the spleen as a purple area seen externally through the skin on the left flank, and make an eight millimeter left flank incision on the skin just above the organ. Next, lift up on the abdominal wall and make a small incision.Allow air into the abdominal cavity so that the internal organs move away from the incision site. Then enlarge the abdominal wall incision to approximately five millimeters. The steps demonstrated for injection are critical for the procedure.Leaking of tumor cells during injection can lead to extra-hepatic metastases. Injection should be performed slowly, over 30 to 60 seconds, with no more than 1.5 million cells to prevent portal venous thrombosis. Expose the spleen by using cotton swab to gently apply pressure around the incision.If needed, the pancreatic fat can be gently manipulated to help with exposure, so as not to injure the spleen. Using a one milliliter syringe with a 27-gauge needle, over a period of 30 to 60 seconds, inject 100 microliters of cells into the tip of the exposed spleen. At the end of the injection, place a micro-clip on the spleen prior to removing the needle, to prevent leakage of the injected cell suspension, and leave it in place for five minutes.Five minutes post-injection, use a hand-held cautery device to perform a splenectomy for hemostasis. Begin with the splenic hilum on the anterior side by pre-coagulating the proximal side of the vessels with adjacent fat tissue. Splenic bleeding can occur at the injection site.The micro-clip is applied to the spleen to prevent tumor cell leakage and to control splenic bleeding. Methods of hemostasis can include cautery, holding pressure for three to five minutes, and suture ligation if necessary. Close the abdominal wall using a 5.0 absorbable braided suture and a single horizontal stitch.Then, use a 4.0 non-absorbable mono-filament suture to close the skin, also with a single horizontal stitch. Monitor the animal post-operatively, according to the text protocol. To perform bioluminescent imaging, after anesthetizing mice according to the text protocol, intraperitoneally inject the animals with 150 microliters of previously prepared firefly luciferin.Place the mice in an IVIS in a supine position with spacers in between the animals to minimize the signal to adjacent mice. Three minutes after luciferin injection, after initializing the IVIS, select luminescent, one second exposure time, and medium binning. Click the Acquire button to start imaging, ensuring that imaging is consistently performed three minutes after luciferin injection.After the image is acquired, and an image window and tool palette appear, click ROI tools and select a number from one to five from the circle icon that corresponds to the number of mice imaged. TO define ROIs, circle the signal area of the luminescent signal on the abdominal cavity of the mouse, and then click measurements of the ROI as an arbitrary unit of radiance. Include an additional ROI circle for the background.Four weeks post-injection, to carry out diffuse luminescent imaging tomography, or DLIT, after initializing the IVIS, select Imaging Wizard, Bioluminescence, and click Next. Select DLIT and click Next. Select Firefly Probes, and click Next.Select Mouse for Image Subject, Auto for Exposure Parameter C-13 Centimeter for Field of View, and 0.5 Centimeter as Subject Type, and click Next. Then click the Acquire button to start imaging. After acquiring an image, select the DLIT 3D Reconstruction tab, in the Analyze tab, and click Reconstruct to generate the 3D reconstruction image.Click Tools and 3D Animation. Then, in the 3D Animation window, select Spin CCW on Y-Axis. Click Record and Save to save the file into a mov format file.To carry out ex vivo fluorescent imaging, after harvesting and dissecting livers from mice according to the text protocol, measure the fluorescent intensities for each tumor colony using the IVIS. By selecting Fluorescence, four seconds Exposure Time, Medium Binning, 2 for F/Stop, 535 for the excitation filter, and 580 for the emission filter. Click the Acquire button to start imaging.After acquiring the image, locate the Image window in Tool Palette. Click ROI Tools, and select 1 from the circle icon. To define ROIs, circle the signal area of individual tumor colonies on the image.Acquire an additional ROI circle of the background, and then click Measurements of the ROI as an arbitrary unit of radiance. Acquire an additional ROI circle of the background. Finally, carry out calculations according to the text protocol.As shown here by bioluminescence, three weeks after spleen injection, HCT116 L2T clones P1 and P2 had a larger tumor burden, as compared to clones O1 and O2.These data indicated that clones O1 and O2 can mimic oligometastatic disease, while P1 and P2 represent aggressive wide-spread metastatic colonization of the liver. The quantification of ex vivo fluorescence of the liver, as of four weeks after spleen injection, confirmed that P1 and P2 had higher fluorescent intensities compared with O1 and O2 livers. Fluorescent labeling of tumor clones identified the number and sizes of individual metastatic colonies in the liver.Overall, ex vivo fluorescent images were consistent with macroscopic findings, but small colonies hidden under the surface were better detected by fluorescent imaging. As depicted here, the numbers and sizes of metastatic colonies were counted and measured in each liver, and ex vivo fluorescence imaging was done on each metastatic colony for each monoclone. As shown in these graphs, the total number of metastases in P1 was higher as compared to P2, O1, and O2, while the average size of individual colonies was larger in P2 than in P1, O1, and O2.Our model provides an approach to identifying new persuasion genes associated with molecular heterogeneity of metastasis.These genes can be used as targets for new approaches to metastasis treatment. Our model can be also used for validation of different protein-coding and non-coding genes which emerge from current large clinical databases but are not yet functionally defined in the context of metastatic disease. Generally, individuals new to this method will struggle, because of the delicacy of the animal surgery.However, with practice, good results are achievable in a short amount of time. While attempting this procedure, it's important to remember to follow animal surgery guidelines, carefully control bleeding, and to prevent the leakage of tumor cell suspension.

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Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

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 ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

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.

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 ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.