We thank Emily Norton for assisting with viral infections and critical reading of the manuscript. We also thank Ryan Kanai for help with acquiring images of the lungs and Kate E. Tubbesing for help with image analysis of the green metastases in the lungs. We thank the animal research facility staff for their support and for assistance in the preparation of this video. This work was supported by a Susan G. Komen Career Catalyst Grant that awarded to J.M.L. (#CCR17477184).

The authors have nothing to disclose.

Critical steps of the method 
It is critical to optimize the number of cells injected (step 3) for a given cell line and mouse strain as this can greatly influence the number of metastases that form and the length of the experiment. If too many cells are injected or the metastases grow for too long, the metastases may be difficult to count making the effects of the genetic manipulation difficult to assess. However, if too few cells are injected, few or no metastases may form. Thus, a preliminary exp...

Cancer remains the second leading cause of death worldwide1 and metastasis is responsible for the majority of these deaths2,3. However, a limited understanding of the molecular mechanisms that govern metastatic colonization and subsequent growth has hindered the development of effective treatments for metastatic disease. The identification of novel therapeutic targets requires an assay to test how perturbed expression or function of a candidate gene influences metastasis formation and growth. While autochthonous mouse models have their advantages, they are time-consuming and expensive to generate, making them more suited for target validation rather than target discovery. Transplant model systems in which the candidate gene is perturbed in cancer cells in vitro and then effects on metastatic potential are assessed in vivo, are less expensive and higher throughput than autochthonous models. In addition, viral vectors for stable delivery of RNAi, CRISPR/CAS9, and transgenes are widely available, making it relatively easy to perturb virtually any gene or genes of interest in a cancer cell lines. This approach can also be used to assay the role of candidate genes in metastatic colonization and growth in human cancer cell lines by transplanting the cells into immunocompromised or humanized mice.
The two types of assays used to test metastasis formation by transplanted cancer cells in vivo are spontaneous metastasis assays and experimental metastasis assays. In spontaneous metastasis assays4,5, cancer cells are injected into mice, allowed to form a primary tumor, and then spontaneous metastasis formation and subsequent growth are assayed. The strength of this model is that the cells must complete all steps of the metastatic process in order to form metastatic tumors. However, many cancer cell lines do not metastasize efficiently in spontaneous metastasis models, and any manipulation of the cells that impacts primary tumor growth can confound the results of the metastasis assay. Experimental metastasis assays, in which cancer cells are injected directly into circulation, are used to avoid these pitfalls. Common experimental metastasis assays include the tail vein injection6,7,8 (and demonstrated here), intracardiac injection9, and portal vein injection10.
The purpose of the protocol presented here is to provide an in vivo experimental metastasis assay that allows a researcher to monitor metastasis formation and growth in real time, as well as to quantify end point metastasis number and size in the lungs of the same mouse. To accomplish this, traditional experimental tail vein metastasis assays6,7,8 are combined with live animal imaging, using an in vivo imaging device9,11,12,13,14. Tumor cells stably expressing both luciferase and a fluorescent protein are injected into mice via the lateral tail vein and then the in vivo imaging device is used to measure changes in metastatic burden in the lungs over time (Figure 1). However, the in vivo live animal imaging device cannot distinguish or measure the size of individual metastases. Thus, at the end of the experiment, a fluorescent stereomicroscope is used to count the number and measure the size of the fluorescent metastases in the lungs without the need for sectioning and histology or immunohistochemistry (Figure 1). This protocol can be used to test how altering the expression or function of a candidate gene influences metastasis formation and growth. Potential therapeutic compounds such as small molecules or function blocking antibodies can also be tested.
To demonstrate this approach, we first performed a proof of concept experiment in which the essential replication factor, replication protein A3 (RPA3) is knocked down in metastatic mouse breast cancer cells. We show that mice injected with RPA3 knockdown cells have significantly less metastatic burden at every time point compared to mice injected with control cells. Analysis of the metastasis-containing lungs shows that this reduced metastatic burden is the result of significantly reduced metastatic colonization and impaired growth of the metastases that form. To further demonstrate this technique, we tested whether simultaneous knock down of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) impairs metastatic colonization or subsequent growth. YAP and TAZ are two related transcriptional co-activators that are the critical downstream effectors of the Hippo Pathway. We15,16 and others have implicated YAP and TAZ in metastasis (reviewed in17,18,19), suggesting that these proteins are good therapeutic targets. Consistently, we found that mice injected with YAP/TAZ knockdown cells had significantly reduced metastatic burden. Analysis of the lungs showed that the YAP/TAZ knockdown cells formed many fewer metastases and that the metastases that did form were smaller. These experiments demonstrate how experimental metastasis assays allow a researcher to quickly and inexpensively test the role of a candidate gene in metastasis formation and growth. They further show how the combined use of live animal imaging and fluorescent quantification of metastases in whole lungs allows the researcher to better understand the steps during metastatic colonization.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% SDS-PAGE Gel</td>
			<td></td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>2.5% Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td>Trypsin for tissue culture</td>
		</tr>
		<tr>
			<td>96 well flat bottom white assay plate</td>
			<td>Corning</td>
			<td>3922</td>
			<td>For measuring luciferase and renilla signal in cultured cells</td>
		</tr>
		<tr>
			<td>Alcohol wipes</td>
			<td></td>
			<td></td>
			<td>For sterolizing the injection site before tail vein injecitons</td>
		</tr>
		<tr>
			<td>BALB/C mice (female, 6 weeks)</td>
			<td>Taconic</td>
			<td>BALB-F</td>
			<td>For tail vein metastatic colonization and burden assays</td>
		</tr>
		<tr>
			<td>BSA regular</td>
			<td>VWR Ameresco</td>
			<td>97061-416</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Cell lysis buffer</td>
			<td>Cell Signaling</td>
			<td></td>
			<td>For collecting protien samples</td>
		</tr>
		<tr>
			<td>Celltreat Syringe Filters, PES 30mm, 0.45 &#956;m</td>
			<td>Celltreat</td>
			<td>40-229749-CS</td>
			<td>For filtering viral supernatant</td>
		</tr>
		<tr>
			<td>CO2 and euthanasia chamber</td>
			<td></td>
			<td></td>
			<td>For euthanasing the mice</td>
		</tr>
		<tr>
			<td>Dual-luciferase reporter assay kit</td>
			<td>Promega</td>
			<td>E1960</td>
			<td>For measuring luciferase and renilla signal in cultured cells</td>
		</tr>
		<tr>
			<td>Dulbecco&39;s phosphate buffered saline</td>
			<td>Himedia</td>
			<td>TS1006</td>
			<td>For PBS</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>VWR</td>
			<td>97061-406</td>
			<td>Used to dilute trypsin for tissue culture</td>
		</tr>
		<tr>
			<td>FBS 100% US origin</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Fujifilm LAS-3000 gel imager</td>
			<td>Fujifilm</td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>GAPDH(14C10) Rabibit mAb</td>
			<td>Cell Signaling</td>
			<td>2118</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) Secondary Antibody, HRP conjugate</td>
			<td>Thermo Scientific</td>
			<td>31460</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Human embryonic kidney cells, HEK-293FT</td>
			<td>Invitrogen</td>
			<td>R70007</td>
			<td>Cell line used for packging virus</td>
		</tr>
		<tr>
			<td>HyClone DMEM/High clucose</td>
			<td>GE Healthcare life sciences</td>
			<td>SH30243.01</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Hygromycin B, Ultra Pure Grade</td>
			<td>VWR Ameresco</td>
			<td>97064-810</td>
			<td>For antibiotic selection of infected cells</td>
		</tr>
		<tr>
			<td>I3-P/i3 Multi-Mode Microplate/EA</td>
			<td>Molecular devices</td>
			<td></td>
			<td>For measuring luciferase and renilla signal in cultured cells</td>
		</tr>
		<tr>
			<td>Imagej</td>
			<td></td>
			<td></td>
			<td>Used for image analysis of lung metastases: threshold set to 25 &#38; 100</td>
		</tr>
		<tr>
			<td>Immuno-Blot PVDF Membrane</td>
			<td>Biorad</td>
			<td>1620177</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td>For mouse anesthesia</td>
		</tr>
		<tr>
			<td>IVIS Lumina XRMS In Vivo Imaging System (in vivo live animal imaging device)</td>
			<td>PerkinElmer</td>
			<td>CLS136340</td>
			<td>For in vivo imaging of metastatic burden</td>
		</tr>
		<tr>
			<td>Leica M205 FA &#38; Lecia DCF3000 G (GFP and bright field filters)</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Microscope and camera for visualing, counting and taking pcitures of metastases in the lungs; 10X magnifacation, 3.5 sec exposure, 1.4 gain</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Life technologies</td>
			<td>L3000008</td>
			<td>For YAP/TAZ-TEAD reporter transfection</td>
		</tr>
		<tr>
			<td>Living Image 3.2 (image software program)</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Software for IVIS</td>
		</tr>
		<tr>
			<td>Mouse breast cancer cells, 4T1</td>
			<td>Karmanos Cancer Institute</td>
			<td>Aslakson, CJ et al.,1992</td>
			<td>Mouse metastatic breast cance cell line</td>
		</tr>
		<tr>
			<td>Multi-Gauge version 3.0</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Software for quantifying western blot band intensity</td>
		</tr>
		<tr>
			<td>Opti-MEM (transfection buffer)</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td>For packaging virus and transfection</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Pierce BCA protein assay kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>For quantifying protein concentration</td>
		</tr>
		<tr>
			<td>Pierce Phosphatase Inhibitor Mini Tablets</td>
			<td>Thermo Scientific</td>
			<td>A32957</td>
			<td>Added to cell lysis buffer</td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor Mini Tablets</td>
			<td>Thermo Scientific</td>
			<td>A32953</td>
			<td>Added to cell lysis buffer</td>
		</tr>
		<tr>
			<td>Polybrene (hexadimethrine bromide)</td>
			<td>Sigma-Aldrich</td>
			<td>45-H9268</td>
			<td>For infection</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>45-P7255</td>
			<td>For antibiotic selection of infected cells</td>
		</tr>
		<tr>
			<td>Rodent restrainer</td>
			<td></td>
			<td></td>
			<td>For restraining mice during tail vein injeciton</td>
		</tr>
		<tr>
			<td>SDS-PAGE running buffer</td>
			<td></td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>TAZ (V3886) Antibody</td>
			<td>Cell Signaling</td>
			<td>4883</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>TBST buffer</td>
			<td></td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>TC20 automated cell counter</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For counting cells</td>
		</tr>
		<tr>
			<td>Vectors</td>
			<td></td>
			<td></td>
			<td>See Table 1 for complete list of vectors</td>
		</tr>
		<tr>
			<td>VWR Inverted Fluorescence Microscope</td>
			<td>VWR</td>
			<td>89404-464</td>
			<td>For visualizing fluorescence in ZSGreen labeled cells</td>
		</tr>
		<tr>
			<td>Western transfer buffer</td>
			<td></td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>XenoLight D-Luciferin K+ Salt</td>
			<td>PerkinElmer</td>
			<td>122799</td>
			<td>Substrate injected into mice for in vivo bioluminescent IVIS images</td>
		</tr>
		<tr>
			<td>X-tremeGENE 9 DNA transfection reagent (lipid solution for transfection)</td>
			<td>Roche</td>
			<td>6365787001</td>
			<td>For packaging virus</td>
		</tr>
		<tr>
			<td>YAP (D8H1X) XP Rabbit mAb</td>
			<td>Cell Signaling</td>
			<td>14074</td>
			<td>For western blot</td>
		</tr>
	</tbody>
</table>

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.

To demonstrate the above approach, we performed a proof of concept experiment in which a critical replication factor, RPA3 was knocked down in a metastatic mouse mammary carcinoma cell line (4T122). While the protocol describes labeling the cells with both luciferase and fluorescent proteins prior to genetic manipulation, we used a modified approach because THE RNAi vectors also deliver ZsGreen (Figure 2A). First, 4T1 cells were stably transduced with...

Watch this Scientific Journal Video about Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs at JoVE.co

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

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 ...

combined-use-tail-vein-metastasis-assays-real-time-vivo-imaging-to

Research

JoVE Journal

Cancer Research

10.7K Views.  Albany Medical College. 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.

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

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

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

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

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

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 ...

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 ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

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 ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.