We thank Christine F. Wogan, MS, ELS, of MD Anderson&#8217;s Division of Radiation Oncology for scientific editing of the manuscript, and Carol M. Johnston from MD Anderson&#8217;s Division of Surgery Histology Core for help with hematoxylin and eosin staining. We are thankful to the Veterinary Medicine and Surgery Core at MD Anderson for their support for the animal studies. This work was supported by the following grants: Susan G. Komen Career Catalyst Research grant (CCR16377813 to BGD), American Cancer Society Research Scholar grant (RSG-19&#8211;126&#8211;01 to BGD), and the State of Texas Rare and Aggressive Breast Cancer Research Program. Also supported in part by Cancer Center Support (Core) Grant P30 CA016672 from the National Cancer Institute, National Institutes of Health, to The University of Texas MD Anderson Cancer Center.

The method described here has been approved by the Institutional Animal Care and Use Committee (IACUC) of the MD Anderson Cancer Center and complies with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The schematic workflow, with all steps included, is presented as Figure 1.
1. Cell preparation
NOTE: The MDA-IBC3 (ER-/PR-/HER2+) cell line, generated in Dr. Woodward&#39;s lab24, was stably transduced with a luciferase-green fluorescent protein (Luc-GFP) plasmid.
<ol>
	<li>Culture transduced cells in Ham&#39;s F-12 media supplemented with 10% fetal bovine serum (FBS), 1 &#181;g/mL hydrocortisone, 5 &#181;g/mL insulin, and 1% antibiotic-antimitotic.</li>
	<li>Plate MDA-IBC3-Luc-GFP cells at 37 &#176;C and 5% CO2 in a T-75 flask until confluent. Change media every 3 days before cells are confluent enough to passage.</li>
	<li>Harvest cells by removing media, washing each flask with 10 mL of 1x Dulbecco&#39;s (i.e., calcium- and magnesium-free) phosphate-buffered saline (DPBS), and adding 2 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Incubate for 3-5 min at 37 &#176;C until cells detach.</li>
	<li>Add 5 mL of complete media to the flask to collect the cells and transfer the entire contents to a 15 mL centrifuge tube (optimized to 50 mL centrifuge tube depending on the total number of cells needed). Centrifuge at 290 x g for 5 min to pellet cells. 
	NOTE: Complete media is Ham&#39;s F-12 media supplemented with 10% fetal bovine serum (FBS), 1 &#181;g/mL hydrocortisone, 5 &#181;g/mL insulin, and 1% antibiotic-antimitotic.</li>
	<li>Discard the supernatant. Then wash the cells with 10 mL of 1x DPBS. Centrifuge at 290 x g for 5 min to pellet the cells and discard the supernatant carefully. Resuspend the cells with 1 mL of fresh 1x DPBS and mix well by pipetting up and down.</li>
	<li>To make single-cell suspensions, filter cells through 40 &#181;m sterile cell strainers.</li>
	<li>Calculate cell density by using an automated cell counter. 
	NOTE: To reduce errors in counting, create different cell dilutions and count them separately, and calculate the average value to determine the concentration of cells (number of cells/mL).
	<ol>
		<li>Collect 2 &#181;L of cell suspension + 8 &#181;L of 1x DPBS to make a 1:5 dilution sample.</li>
		<li>Collect 1 &#181;L of cell suspension + 9 &#181;L of 1x DPBS to make a 1:10 dilution sample.</li>
		<li>Add 10 &#181;L of 0.4% trypan blue stain. Mix the sample mixture well by pipetting it up and down a few times.</li>
		<li>Gently pipette 10 &#181;L of the sample into the half-moon-shaped sample loading area of the counting chamber slides. Make sure there are no bubbles inside; wait for 30 s to allow the cells to settle in the chamber before counting.</li>
	</ol>
	</li>
	<li>Dilute the cells with&#160;1x DPBS to a density of 5 x 105 or 1 x 106 cells per 100 &#181;L. Transfer cell suspension into 1.7&#160;mL microcentrifuge tubes for tail-vein injection. Place cells on ice until injection to maintain viability.</li>
</ol>
2. Tail vein injection
<ol>
	<li>Use 4- to 6-week-old female athymic SCID/Beige mice.</li>
	<li>Prepare a 30 G syringe to draw 100 &#181;L of cell suspension. Remove all air bubbles.</li>
	<li>Place mouse in a rodent restrainer (diameter about 1 inch) to facilitate tail-vein injection and prevent injuries from movement during the injection process.</li>
	<li>Use alcohol cotton pads (made with 70% ethanol) to gently wipe the mouse&#39;s tail 3-4 times and hold for 5-10 s to make the tail vein more clearly visible.</li>
	<li>Insert the syringe into the tail of the mouse at an angle of 15-30&#176;. Slowly push the cell suspension into the tail vein.</li>
	<li>Remove the syringe gently and use the cotton pad to hold the tail for few seconds to help stop any bleeding.</li>
	<li>Return the mice to their cages and check them 2-4 h after injection to ensure that no adverse effects occur.</li>
</ol>
3. Evaluation of brain metastasis burden
<ol>
	<li>Prepare diluted D-luciferin solution. 
	&#8203;NOTE: Stock concentration is 47.6 mM (15.15 mg/mL), and the concentration for use is 1.515 mg/mL.
	<ol>
		<li>Add 5 mL of 1x DPBS to the D-luciferin bottle.</li>
		<li>Place 2.5 mL of the solution into each of two 50 mL conical tubes.</li>
		<li>Add another 5 mL of 1x DPBS to the D-luciferin bottle again to rinse it. Put 2.5 mL into each 50 mL conical tube.</li>
		<li>Place another 5 mL of 1x DPBS into the bottle and rinse it again.Repeat the rinsing process twice.</li>
		<li>Add 1x DPBS to each 50 mL tube to a total of 66 mL (about 33 mL per tube). There should be about 33 mL of the solution in each tube.</li>
		<li>Mix the tubes well and then combine both into a sterile filtration unit (150 mL PES filter 0.45 &#181;m).</li>
		<li>Filter the solution and then aliquot the filtered solution into sterile amber 1.7&#160;mL microtubes.</li>
	</ol>
	</li>
	<li>Detecting brain metastases by luciferase imaging.
	<ol>
		<li>Thaw D-luciferin by keeping on ice, and vortex before injection into the mice.</li>
		<li>Clean the area of injection with alcohol cotton pads and inject 100 &#181;L of D-luciferin per mouse intraperitoneally by using a 0.5 mL insulin syringe, one syringe for each cage.</li>
		<li>After 10 min, anesthetize the mice with isoflurane (2% O2- 2.5% isoflurane) for 5 min using the veterinary vaporizer connected to the small animal induction chamber.</li>
		<li>Turn on the in vivo imaging system. While the mice from the first cage are under anesthesia, inject the D-luciferin into the mice in the next cage.</li>
		<li>Put mice into the in vivo imaging system machine and obtain ventral and dorsal images. Select an exposure time of 2 min, and in the field view choose option E (whole body). Press Acquire. Next, save the image (Figure 2). 
		&#8203;NOTE: If the image shows saturation of luminescence, reduce the exposure time.</li>
	</ol>
	</li>
	<li>Detecting brain metastases by GFP imaging.
	<ol>
		<li>Brain tissue preparation.
		<ol>
			<li>About 8-10 weeks post injection of cells or when the mice are moribund due to brain metastasis burden, euthanize the mice by inhalation of an isoflurane overdose followed by cervical dislocation, in accordance with protocols approved by the IACUC.</li>
			<li>After euthanasia with isoflurane followed by cervical dislocation, spray the mice with a 70% ethanol solution and remove the fur from the head area and ears with scissors. Next, make a cut in the cervical area of the neck (being careful to not decapitate the mouse). Cut the skull and pull it out. Next, carefully remove the brain.</li>
			<li>Place the collected brains into tissue cassettes.</li>
			<li>Transfer the cassettes to a container with cold 1x DPBS. 
			&#8203;NOTE: Brain samples should not be kept in 1x DPBS for more than 1 h. If several brain samples are to be collected at the same time, euthanize only a few mice at a time (10 per round).</li>
		</ol>
		</li>
		<li>Stereomicroscopic imaging.
		<ol>
			<li>Transfer the whole brain onto a 100 cm tissue dish lid.</li>
			<li>Turn on the stereomicroscope and the UV light, at position 3 with lens 0.5x, to allow visualization of the whole brain as shown in Figure 3A (red square).</li>
			<li>Press Live view (Figure 3A, green square).</li>
			<li>Focus the view while the brain is in the ventral position.</li>
			<li>Select GFP (if cells are GFP-labeled) or TXRED (if cells are RFP-labeled) (Figure 3A, yellow square) in the software, select the proper filter on the microscope, and take a picture. 
			&#8203;NOTE: Keep the SOLA light source setting at about 30%; if the GFP is too bright, reduce until the proper signal is observed.</li>
			<li>Without moving the sample, turn on the bright light and select BF (Figure 3A, yellow square). Take a picture.</li>
			<li>Repeat the previous steps with the brain in the dorsal position. 
			&#8203;NOTE: Depending on the size, the same GFP positive brain metastasis lesion can appear in both ventral and dorsal positions. In such a case, avoid duplicate quantification of the same lesion. Save the images with a TIFF extension (which keeps all the information in the picture file).</li>
			<li>Save the images with a TIFF extension (which keeps all the information in the picture file).</li>
			<li>Repeat steps for all samples.</li>
			<li>Convert the TIFF images into JPEG so that the files can be opened with any computer that does not have the associated software installed (File | Import/Export | convert files).</li>
			<li>To merge the fluorescent image with the bright field image, open both images and go to File | Merge channels. Select the proper components, green for GFP and brightfield for BF, and press Ok. Save the merged image.</li>
			<li>To quantify brain tumor area, use the fluorescent image. Select Measure | Manual Measurement | Area. Select automatic selection (Figure 3B, green square), and move the arrow to the GFP-positive area and click to automatically create a selection. Click again to confirm the selection and then all measurements will show (Figure 3B, red square). If more than one GFP-positive area is present, repeat the procedure.</li>
		</ol>
		</li>
		<li>After brain images are obtained, proceed to routine tissue fixation protocols using 10% neutral buffered formalin.
		<ol>
			<li>Once the brains are fixed, proceed to standard histological sectioning followed by hematoxylin and eosin staining to confirm the presence of brain metastasis and immunohistochemical staining to detect selected markers. 
			NOTE: Immunocytochemistry was performed at the Pathology Core Laboratory at UT MD Anderson Cancer Center that has standardized protocol for immunohistochemical staining of known markers.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

The protocol includes several critical steps. Cells should be kept on ice for no longer than 1 hour to maintain viability. Alcohol cotton pads should be used to wipe the tails of the mice before injection, with care taken to not wipe too hard or too often to avoid damaging the tail skin. Ensure that no air bubbles are present in the cell suspension, to prevent mice from dying from blood vessel emboli. Maintain the angle of injection at 45&#176; or less to avoid piercing the blood vessel in the tails and insert at least 1...

Brain metastasis is a common and deadly complication of systemic malignancies. Most brain metastases originate from primary tumors of the lung, breast or skin, which collectively account for 67-80% of cases1,2. Estimates of the incidence of brain metastasis vary between 100,000 to 240,000 cases, and these numbers may be underestimates because autopsy is rare for patients who died of metastatic cancer3. Patients with brain metastases have a worse prognosis and lower overall survival relative to patients without brain metastases4. Current treatment options for brain metastases are largely palliative and fail to improve survival outcomes for most patients5. Thus, brain metastasis remains a challenge, and the need remains pressing to better understand the mechanisms of brain metastasis progression to develop more effective therapies.
The use of experimental models has provided important insights into specific mechanisms of breast cancer metastatic progression to the brain and allowed evaluation of the efficacy of various therapeutic approaches6,7,8,9,10,11,12,13,14,15,16. However, very few models can accurately and fully recapitulate the intricacies of brain metastasis development. Several experimental in vivo models have been generated via inoculation of cancer cells into mice by different routes of administration, including orthotopic, tail-vein, intracardiac, intracarotid arterial, and intracerebral injections. Each technique has advantages and disadvantages, as reviewed elsewhere3. None of these mouse models, however, can fully replicate the clinical progression of brain metastasis.
Brain metastases are particularly common in patients with inflammatory breast cancer (IBC), a rare but aggressive variant of primary breast cancer. IBC accounts for 1% to 4% of breast cancer cases, but it is responsible for a disproportionate 10% of breast cancer-related deaths in the United States17,18. IBC is known to rapidly metastasize; indeed, one-third of IBC patients have distant metastasis at the time of diagnosis19,20. Specific to brain metastasis, patients with IBC have a higher incidence of brain metastasis than do patients with non-IBC21. Recently, we demonstrated that the MDA-IBC3 cell line, derived from the malignant pleural effusion fluid of a patient with ER-/PR-/HER2+ IBC that recapitulates IBC characteristics in mouse xenografts, has an enhanced propensity to develop brain metastases rather than lung metastases in mice when injected by tail-vein, making this cell line a good model for studying the development of brain metastasis16.
Herein we describe the procedures to generate brain metastasis via tail-vein injection of MDA-IBC3 cells and to evaluate the metastatic burden via stereofluorescent microscopy and luciferase imaging. This method has been used to discover key mediators of breast cancer metastasis to the brain and to test the efficacy of therapeutic interventions16,22,23. The disadvantage of this technique is that it does not recapitulate all the steps in the brain metastatic process. Nevertheless, its major advantages include robustness and reproducibility, involvement of the relevant metastasis biology of intravasation, traversing the lungs and extravasation into the brain, and its relative simplicity in terms of technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L pipette tip filtered</td>
			<td>Genesee Scientific</td>
			<td>23430</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipets</td>
			<td>Genesee Scientific</td>
			<td>12-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Centrifuge tubes 15 mL bulk</td>
			<td>Genesee Scientific</td>
			<td>28103&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160; 500 mL Hams F-12 Medium [+] L-glutamine</td>
			<td>GIBICO Inc. USA</td>
			<td>MT10080CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter (Invitrogen)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>1x DPBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf centufuge 5810R</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GIBICO Inc. USA</td>
			<td>16000044</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Fisherbrand &#160;Sterile Cell Strainers (40 &#956;m)</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-363-547</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>12585014</td>
			<td>5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Invitrogen&#160;Countess Cell Counting Chamber Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-IBC3 cell lines</td>
			<td>MD Anderson Cancer Center</td>
			<td></td>
			<td>Generated by Dr. Woodward&#39;s lab24</td>
		</tr>
		<tr>
			<td>Luciferase&#8211;green fluorescent protein (Luc&#8211;GFP) plasmid</td>
			<td>System Biosciences</td>
			<td>BLIV713PA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes clear sterile 1.7 mL</td>
			<td>Genesee Scientific</td>
			<td>24282S</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus 10 &#181;L Reach Barrier Tip, Low Binding, Racked, Sterile</td>
			<td>Genesee Scientific</td>
			<td>23-401C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Treated Flasks (T75), 250mL, Vent</td>
			<td>Genesee Scientific</td>
			<td>25-209</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%) for use with the Countess Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>Tail vein injection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C.B-17/IcrHsd-Prkdc scid Lyst bg-J - SCID/Beige</td>
			<td>Envigo</td>
			<td>SCID/beige mice</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Insulin Syringe with the BD Ultra-Fine Needle 0.5mL 30Gx1/2&#34; (12.7mm)</td>
			<td>BD</td>
			<td>328466</td>
			<td></td>
		</tr>
		<tr>
			<td>Plas Labs &#160;Broome-Style Rodent Restrainers</td>
			<td>Plas Labs 551BSRR</td>
			<td>01-288-32A</td>
			<td>Order fromThermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Volu SolSupplier Diversity Partner Ethanol 95% SDA (190 Proof)</td>
			<td>Thermo Fisher Scientific</td>
			<td>50420872</td>
			<td>70 % used</td>
		</tr>
		<tr>
			<td>Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Lo-Dose&#160; U-100 Insulin Syringes</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable PES Filter Units 0.45 &#181;m</td>
			<td>Fisherbrand</td>
			<td>FB12566501</td>
			<td>filter system to sterilize the D-luciferin</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Biosynth</td>
			<td>L8220-1g</td>
			<td>stock concentration = 47.6 mM (15.15 mg/mL); use concentration = 1.515 mg/mL</td>
		</tr>
		<tr>
			<td>1.7 mL microtube amber</td>
			<td>Genesee Scientific</td>
			<td>24-282AM</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>NDC-14043-704-06</td>
			<td>Liquid anesthetic for use in anesthetic vaporizer</td>
		</tr>
		<tr>
			<td>IVIS 200&#160;</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>machine for luciferase imaging, up to 5 mice imaging at the same time, with anesthesia machine</td>
		</tr>
		<tr>
			<td>Plastic Containers with Lids&#160;</td>
			<td>Fisherbrand</td>
			<td>02-544-127</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cassettes</td>
			<td>Thermo Scientific</td>
			<td>1000957</td>
			<td></td>
		</tr>
		<tr>
			<td>Webcol Alcohol Prep&#160;</td>
			<td>Covidien</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope AZ100&#160;</td>
			<td>Nikon</td>
			<td>model AZ-STGE</td>
			<td>software NIS-ELEMENT</td>
		</tr>
		<tr>
			<td>Formalin 10%</td>
			<td>Fisher Chemical</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated dishes 100x20 mm</td>
			<td>Genesee Scientific</td>
			<td>25202</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

With the rationale that labeled cells facilitate monitoring and visualization of brain metastasis in preclinical mouse models, we tagged MDA-IBC3 cells with Luc and with GFP to monitor brain metastases and quantify the metastatic burden by using bioluminescence imaging and fluorescent stereomicroscopy. Injection of the labeled MDA-IBC3 cells into the tail veins of immunocompromised SCID/Beige mice resulted in high percentages of mice developing brain metastasis (i.e., 66.7% to 100 %)16,...

Watch this Scientific Journal Video about Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells at JoVE.com

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

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 ...

modeling-brain-metastasis-via-tail-vein-injection-inflammatory-breast

Research

JoVE Journal

Cancer Research

3.2K Views.  The University of Texas MD Anderson Cancer Center. 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.

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

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

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 ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

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 ...

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 ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...