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...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+metastases+in+patients+with+HER2-positive+metastatic+breast+cancer:+incidence,+treatment,+and+survival+in+patients+from+registHER.">Central nervous system metastases in patients with HER2-positive metastatic breast cancer: incidence, treatment, and survival in patients from registHER.</a> Clinical Cancer Research. 17 (14), 4834-4843 (2011).</li><li>Valiente, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+landscape+of+brain+metastasis.">The evolving landscape of brain metastasis.</a> Trends in Cancer. 4 (3), 176-196 (2018).</li><li>Bos, P. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+upregulate+survival+genes+in+tumor+cells+and+induce+protection+from+chemotherapy.">Astrocytes upregulate survival genes in tumor cells and induce protection from chemotherapy.</a> Neoplasia. 13 (3), 286-298 (2011).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRC+family+kinases+as+novel+therapeutic+targets+to+treat+breast+cancer+brain+metastases.">SRC family kinases as novel therapeutic targets to treat breast cancer brain metastases.</a> Cancer Research. 73 (18), 5764-5774 (2013).</li><li>Valiente, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serpins+promote+cancer+cell+survival+and+vascular+co-option+in+brain+metastasis.">Serpins promote cancer cell survival and vascular co-option in brain metastasis.</a> Cell. 156 (5), 1002-1016 (2014).</li><li>Gril, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+lapatinib+on+the+outgrowth+of+metastatic+breast+cancer+cells+to+the+brain.">Effect of lapatinib on the outgrowth of metastatic breast cancer cells to the brain.</a> Journal of the National Cancer Institute. 100 (15), 1092-1103 (2008).</li><li>Gril, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pazopanib+reveals+a+role+for+tumor+cell+B-Raf+in+the+prevention+of+HER2++breast+cancer+brain+metastasis.">Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis.</a> Clinical Cancer Research. 17 (1), 142-153 (2011).</li><li>Palmieri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+prevention+of+experimental+brain+metastases+of+breast+cancer+by+temozolomide+in+an+MGMT-dependent+manner.">Profound prevention of experimental brain metastases of breast cancer by temozolomide in an MGMT-dependent manner.</a> Clinical Cancer Research. 20 (10), 2727-2739 (2014).</li><li>Priego, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+labels+a+subpopulation+of+reactive+astrocytes+required+for+brain+metastasis.">STAT3 labels a subpopulation of reactive astrocytes required for brain metastasis.</a> Nature Medicine. 24 (7), 1024-1035 (2018).</li><li>Debeb, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-141-mediated+regulation+of+brain+metastasis+from+breast+cancer.">miR-141-mediated regulation of brain metastasis from breast cancer.</a> Journal of the National Cancer Institute. 108 (8), (2016).</li><li>Chang, S., Parker, S. L., Pham, T., Buzdar, A. U., Hursting, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+breast+carcinoma+incidence+and+survival:+the+surveillance,+epidemiology,+and+end+results+program+of+the+National+Cancer+Institute,+1975-1992.">Inflammatory breast carcinoma incidence and survival: the surveillance, epidemiology, and end results program of the National Cancer Institute, 1975-1992.</a> Cancer. 82 (12), 2366-2372 (1998).</li><li>Hance, K. W., Anderson, W. F., Devesa, S. S., Young, H. A., Levine, P. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+breast+cancer:+Current+understanding.">Inflammatory breast cancer: Current understanding.</a> Current Opinion in Oncology. 18 (6), 563-571 (2006).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+of+distant+metastases+in+inflammatory+breast+cancer+-+A+large-cohort+retrospective+study.">Pattern of distant metastases in inflammatory breast cancer - A large-cohort retrospective study.</a> Journal of Cancer. 11 (2), 292-300 (2020).</li><li>Uemura, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+CNS+metastases+and+survival+in+patients+with+inflammatory+breast+cancer.">Development of CNS metastases and survival in patients with inflammatory breast cancer.</a> Cancer. 124 (11), 2299-2305 (2018).</li><li>Smith, D. L., Debeb, B. G., Thames, H. D., Woodward, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+modeling+of+micrometastatic+breast+cancer+radiation+dose+response.">Computational modeling of micrometastatic breast cancer radiation dose response.</a> International Journal of Radiation Oncology, Biology, Physics. 96 (1), 179-187 (2016).</li><li>Fukumura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-omic+molecular+profiling+reveals+potentially+targetable+abnormalities+shared+across+multiple+histologies+of+brain+metastasis.">Multi-omic molecular profiling reveals potentially targetable abnormalities shared across multiple histologies of brain metastasis.</a> Acta Neuropathol. , (2021).</li><li>Klopp, A. 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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 ...

We utilized the tail vein method to generate brain metastasis in mouse models using cell lines derived from patients with inflammatory breast cancer, or IBC, a highly aggressive form of primary breast cancer. The generation of brain metastasis in mouse models typically involves intracardiac or intracarotid arterial injections. However, the tail vein method offers advantage over these techniques as it better mimics the steps of brain metastasis colonization, and is relatively straightforward to perform.Doctors Xiaoding Hu and Emilly Villodre, both of whom are instructors in my laboratory, will demonstrate the procedure. To detect brain metastases by GFP imaging eight to 10 weeks after tumor cell injection, disinfect the mouse with 70%ethanol and remove the fur from the head and ears. Cut the cervical area of the neck.Excise and remove the skull to allow the brain to be harvested. Place the brain into a tissue cassette and place the cassette into a container of cold DPBS for no more than one hour. For imaging by stereo microscopy, transfer the brain into a 100-centimeter tissue dish lid under a stereo microscope equipped with a UV light and select the 0.5x objective to allow visualization of the entire brain.Press live view and focus the view while the brain is in the ventral position. Select the appropriate filter in the software and microscope for the fluorescent marker in the cells, and take a picture. Without moving the sample, switch to brightfield imaging and select the brightfield filter.Take a picture. Then image the brain in the dorsal position as demonstrated earlier. To analyze the whole brain images, first convert the image files from TIFF to JPEG so they can be opened with any computer that does not have the associated software.Next, open a pair of brightfield and fluorescent images and select file and merge channels. Select the proper components, click okay, and save the merged image. To quantify the brain tumor area in the fluorescent image, select automatic selection.Move the arrow to the GFP position area and click to create a selection automatically. Then click again to confirm the selection. All the measurements will be shown.If more than one GFP positive area can be observed, repeat the measurement with the subsequent area. Brain metastatic lesions can be detected as early as eight weeks after injection by luciferase imaging and stereo fluorescent microscopy. After imaging, portions of the brain metastases can be formalin fixed and processed for hematoxylin and eosin staining to validate the presence of brain metastasis lesions, and for immunohistochemical staining to detect specific protein markers of interest.When attempting This protocol, it is important to maintain cell viability by keeping the cells on ice for no more than one hour, and to avoid air bubbles in the cell suspension to prevent emboli formation and mouse mortality. Following tail vein injection, we monitor brain metastasis growths and progression with luciferase imaging. After euthanasia, stereo fluorescent microscopy and histology confirmed metastasis lesions.

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

Research

JoVE Journal

Cancer Research

3.1K Görüntüleme.  The University of Texas MD Anderson Cancer Center. İnflamatuar meme kanseri veya IBC'li hastalardan elde edilen hücre dizilerini kullanarak fare modellerinde beyin metastazı oluşturmak için kuyruk damarı yöntemini kullandık. Fare modellerinde beyin metastazı oluşumu tipik olarak intrakardiyak veya intrakarotis arteriyel enjeksiyonları içerir. Bununla birlikte, kuyruk damarı yöntemi, beyin metastazı kolonizasyon adımlarını daha iyi taklit ettiği ve gerçekleştirilmesi nispeten basit olduğu için bu tekniklere göre avantaj...