Representative data was funded through the National Cancer Institute (K22CA218549 to S.T.S). In addition to their assistance in developing the comprehensive analysis method reported herein, we thank The Ohio State University Comprehensive Cancer Center Comparative Pathology and Mouse Phenotyping Shared Resource (Director &#8211; Krista La Perle, DVM, PhD) for histology and immunohistochemistry services and the Pathology Imaging Core for algorithm development and analysis.

Animal use followed University Laboratory Animal Resources (ULAR) regulations under the OSU Institutional Animal Care and Use Committee (IACUC)&#8211;approved protocol 2007A0120-R4 (PI: Dr. Gina Sizemore).
1. Tail-vein injection of breast cancer cells
<ol>
	<li>Preparation of cells and syringe for injection
	<ol>
		<li>Plate an appropriate number of cells based on the number of mice and cell concentration to be used. 
		NOTE: The number of cells injected and time to the development of m...

<ol>
	<li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nature Reviews: Cancer. 2 (8), 563-572 (2002).</li><li>Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+metastasis.">Targeting metastasis.</a> Nature Reviews: Cancer. 16 (4), 201-218 (2016).</li><li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+building+a+framework.">Cancer metastasis: building a framework.</a> Cell. 127 (4), 679-695 (2006).</li><li>Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+mechanistic+insights+and+clinical+challenges.">Tumor metastasis: mechanistic insights and clinical challenges.</a> Nature Medicine. 12 (8), 895-904 (2006).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331 (6024), 1559-1564 (2011).</li><li>Eckhardt, B. L., Francis, P. A., Parker, B. S., Anderson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+discovery+and+development+of+therapies+for+metastatic+breast+cancer.">Strategies for the discovery and development of therapies for metastatic breast cancer.</a> Nature Reviews Drug Discovery. 11 (6), 479-497 (2012).</li><li>Gomez-Cuadrado, L., Tracey, N., Ma, R., Qian, B., Brunton, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+metastasis:+progress+and+prospects.">Mouse models of metastasis: progress and prospects.</a> Disease Models &amp; Mechanisms. 10 (9), 1061-1074 (2017).</li><li>Fantozzi, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+breast+cancer+metastasis.">Mouse models of breast cancer metastasis.</a> Breast Cancer Research. 8 (4), 212 (2006).</li><li>Schoenenberger, C. A., 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=Targeted+c-myc+gene+expression+in+mammary+glands+of+transgenic+mice+induces+mammary+tumours+with+constitutive+milk+protein+gene+transcription.">Targeted c-myc gene expression in mammary glands of transgenic mice induces mammary tumours with constitutive milk protein gene transcription.</a> EMBO Journal. 7 (1), 169-175 (1988).</li><li>Nusse, R., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+tumors+induced+by+the+mouse+mammary+tumor+virus+contain+a+provirus+integrated+in+the+same+region+of+the+host+genome.">Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.</a> Cell. 31 (1), 99-109 (1982).</li><li>Muller, W. J., Sinn, E., Pattengale, P. K., Wallace, R., Leder, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+induction+of+mammary+adenocarcinoma+in+transgenic+mice+bearing+the+activated+c-neu+oncogene.">Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene.</a> Cell. 54 (1), 105-115 (1988).</li><li>Lin, E. Y., 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=Progression+to+malignancy+in+the+polyoma+middle+T+oncoprotein+mouse+breast+cancer+model+provides+a+reliable+model+for+human+diseases.">Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases.</a> American Journal of Pathology. 163 (5), 2113-2126 (2003).</li><li>Green, J. E., 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+C3(1)/SV40+T-antigen+transgenic+mouse+model+of+mammary+cancer:+ductal+epithelial+cell+targeting+with+multistage+progression+to+carcinoma.">The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma.</a> Oncogene. 19 (1), 1020-1027 (2000).</li><li>Iorns, E., 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=A+new+mouse+model+for+the+study+of+human+breast+cancer+metastasis.">A new mouse model for the study of human breast cancer metastasis.</a> PloS One. 7 (10), 47995 (2012).</li><li>Kim, I. S., Baek, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+breast+cancer+metastasis.">Mouse models for breast cancer metastasis.</a> Biochemical and Biophysical Research Communications. 394 (3), 443-447 (2010).</li><li>Mariotto, A. B., Etzioni, R., Hurlbert, M., Penberthy, L., Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+Number+of+Women+Living+with+Metastatic+Breast+Cancer+in+the+United+States.">Estimation of the Number of Women Living with Metastatic Breast Cancer in the United States.</a> Cancer Epidemiology, Biomarkers and Prevention. 26 (6), 809-815 (2017).</li><li>Xiao, W., 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=Risk+factors+and+survival+outcomes+in+patients+with+breast+cancer+and+lung+metastasis:+a+population-based+study.">Risk factors and survival outcomes in patients with breast cancer and lung metastasis: a population-based study.</a> Cancer Medicine. 7 (3), 922-930 (2018).</li><li>Smid, 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=Subtypes+of+breast+cancer+show+preferential+site+of+relapse.">Subtypes of breast cancer show preferential site of relapse.</a> Cancer Research. 68 (9), 3108-3114 (2008).</li><li>Kennecke, H., 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=Metastatic+behavior+of+breast+cancer+subtypes.">Metastatic behavior of breast cancer subtypes.</a> Journal of Clinical Oncology. 28 (20), 3271-3277 (2010).</li><li>Soni, A., 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=Breast+cancer+subtypes+predispose+the+site+of+distant+metastases.">Breast cancer subtypes predispose the site of distant metastases.</a> American Journal of Clinical Pathology. 143 (4), 471-478 (2015).</li><li>Leone, B. A., 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=Prognostic+impact+of+metastatic+pattern+in+stage+IV+breast+cancer+at+initial+diagnosis.">Prognostic impact of metastatic pattern in stage IV breast cancer at initial diagnosis.</a> Breast Cancer Research and Treatment. 161 (3), 537-548 (2017).</li><li>Pei, X. F., 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=Explant-cell+culture+of+primary+mammary+tumors+from+MMTV-c-Myc+transgenic+mice.">Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice.</a> In Vitro Cellular and Developmental Biology: Animal. 40 (1-2), 14-21 (2004).</li><li>Mathsyaraja, H., 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=CSF1-ETS2-induced+microRNA+in+myeloid+cells+promote+metastatic+tumor+growth.">CSF1-ETS2-induced microRNA in myeloid cells promote metastatic tumor growth.</a> Oncogene. 34 (28), 3651-3661 (2015).</li><li>Yang, S., Zhang, J. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+tumor+metastasis.">Mouse models for tumor metastasis.</a> Methods in Molecular Biology. 928, 221-228 (2012).</li><li>La Perle, K. M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Pathologists:+Ultimate+Control+Freaks+Seeking+Validation.">Comparative Pathologists: Ultimate Control Freaks Seeking Validation.</a> Veterinary Pathology. 56 (1), 19-23 (2019).</li><li>Blomberg, O. S., Spagnuolo, L., de Visser, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+of+metastasis:+mechanistic+insights+and+therapeutic+opportunities.">Immune regulation of metastasis: mechanistic insights and therapeutic opportunities.</a> Disease Models &amp; Mechanisms. 11 (10), (2018).</li><li>Gonzalez, H., Hagerling, C., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+immune+system+in+cancer:+from+tumor+initiation+to+metastatic+progression.">Roles of the immune system in cancer: from tumor initiation to metastatic progression.</a> Genes and Development. 32 (19-20), 1267-1284 (2018).</li><li>Borowsky, A. 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=Syngeneic+mouse+mammary+carcinoma+cell+lines:+two+closely+related+cell+lines+with+divergent+metastatic+behavior.">Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior.</a> Clinical and Experimental Metastasis. 22 (1), 47-59 (2005).</li><li>Yang, Y., 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=Immunocompetent+mouse+allograft+models+for+development+of+therapies+to+target+breast+cancer+metastasis.">Immunocompetent mouse allograft models for development of therapies to target breast cancer metastasis.</a> Oncotarget. 8 (19), 30621-30643 (2017).</li><li>Resch, M., Neels, T., Tichy, A., Palme, R., Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+assessment+of+tail-vein+injection+in+mice+using+a+modified+anaesthesia+induction+chamber+versus+a+common+restrainer+without+anaesthesia.">Impact assessment of tail-vein injection in mice using a modified anaesthesia induction chamber versus a common restrainer without anaesthesia.</a> Laboratory Animals. 53 (2), 190-201 (2019).</li><li>Rashid, O. 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=Is+tail+vein+injection+a+relevant+breast+cancer+lung+metastasis+model.">Is tail vein injection a relevant breast cancer lung metastasis model.</a> Journal of Thoracic Disease. 5 (4), 385-392 (2013).</li><li>Goodale, D., Phay, C., Postenka, C. O., Keeney, M., Allan, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+tumor+cell+dissemination+patterns+in+preclinical+models+of+cancer+metastasis+using+flow+cytometry+and+laser+scanning+cytometry.">Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry.</a> Cytometry Part A. 75 (4), 344-355 (2009).</li><li>Goddard, E. T., Fischer, J., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Portal+Vein+Injection+Model+to+Study+Liver+Metastasis+of+Breast+Cancer.">A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.</a> Journal of Visualized Experiments. (118), (2016).</li><li>Wright, L. E., 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=Murine+models+of+breast+cancer+bone+metastasis.">Murine models of breast cancer bone metastasis.</a> BoneKEy Reports. 5, 804 (2016).</li><li>Simmons, J. 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=Animal+Models+of+Bone+Metastasis.">Animal Models of Bone Metastasis.</a> Veterinary Pathology. 52 (5), 827-841 (2015).</li><li>Liu, 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=Improving+orthotopic+mouse+models+of+patient-derived+breast+cancer+brain+metastases+by+a+modified+intracarotid+injection+method.">Improving orthotopic mouse models of patient-derived breast cancer brain metastases by a modified intracarotid injection method.</a> Scientific Reports. 9 (1), 622 (2019).</li><li>Kodack, D. P., Askoxylakis, V., Ferraro, G. B., Fukumura, D., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+strategies+for+treating+brain+metastases+from+breast+cancer.">Emerging strategies for treating brain metastases from breast cancer.</a> Cancer Cell. 27 (2), 163-175 (2015).</li><li>Brown, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Stereology+Applications+for+the+Pathologist.">Practical Stereology Applications for the Pathologist.</a> Veterinary Pathology. 54 (3), 358-368 (2017).</li><li>Aeffner, F., 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=Digital+Microscopy,+Image+Analysis,+and+Virtual+Slide+Repository.">Digital Microscopy, Image Analysis, and Virtual Slide Repository.</a> Institute for Laboratory Animal Research Journal. 59 (1), 66-79 (2018).</li></ol>

As researchers continue to use intravenous injection of tumor cells as an experimental model for metastasis, standard practices to analyze the resulting metastatic tumor burden are lacking. In some cases, significant differences in metastatic tumor burden upon manipulation of particular cell lines and/or use of chemical compounds can be observed macroscopically. However, in other instances, subtle differences in metastatic seeding and growth may be overlooked or misinterpreted without thorough pathological analysis. This...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61270/pathological-analysis-lung-metastasis-following-lateral-tail-vein">Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells</a>. The author list was updated.
The author list was updated from:
Katie A. Thies1,&#160;Sue E. Knoblaugh2, and Steven T. Sizemore1 
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 
2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University
to:
Katie A. Thies1, Sarah Steck1, Sue E. Knoblaugh2, and Steven T. Sizemore1 
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 
2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61270/pathological-analysis-lung-metastasis-following-lateral-tail-vein">Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells</a>. The author list was updated.

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

Despite being a highly complex and inefficient process1, metastasis is a significant contributor to the morbidity and mortality of cancer patients2. In fact, most cancer-related deaths are attributed to metastatic spread of disease3,4. In order for tumor cells to successfully metastasize, they must detach from the primary site, invade through adjoining stroma, intravasate into blood circulation or lymphatics, travel to the capillary bed of a secondary site, extravasate into the secondary tissue, and proliferate or grow to form metastatic lesions5. The use of mouse models has been critical to furthering the understanding of the molecular mechanisms responsible for metastatic seeding and growth6,7. Herein, we focus on breast cancer metastasis, for which both genetically modified mouse models as well as methods of transplantation are often used &#8211; each with their own set of advantages and limitations.
Genetically engineered mammary tumor models make use of mammary gland specific promoters, including MMTV-LTR (mouse mammary tumor virus long terminal repeat)&#160;and WAP (Whey Acidic Protein), to drive expression of transgenes in the mammary epithelium8. Oncogenes including polyoma middle T antigen (PyMT), ErbB2/Neu, c-Myc, Wnt-1, and simian virus 40 (SV40) have been expressed in this manner9,10,11,12,13, and while these genetic models are useful for studying primary tumor initiation and progression, few readily metastasize to distant organs. Furthermore, these genetic mouse models are often more time and cost prohibitive than spontaneous or experimental metastasis models. Given the limitation of most genetically engineered mammary tumor models to study metastasis, transplantation techniques have become&#160;attractive methods&#160;to study this complex process. This includes orthotopic, tail-vein, intracardiac, and intracranial injection of suitable cell lines.
Although several breast cancer cell lines readily metastasize following orthotopic injection into the mammary fat pad14,15, the consistency and reproducibility of metastatic tumor burden can be a challenge, and the duration of such studies can be on the order of several months. For evaluating lung metastasis, in particular, intravenous injection into the tail-vein is often a more reproducible and time-effective method with metastatic spread typically occurring within the span of a few weeks. However, since the intravenous injection model&#160;bypasses initial steps of the metastatic cascade, care must be taken in interpreting the results of these studies. In this demonstration, we show tail-vein injection of mammary tumor cells along with an accurate and comprehensive method of analysis.
Even though the research community has made significant progress in understanding the complex process of breast cancer metastasis, it is estimated that over 150,000 women currently have metastatic breast cancer16. Of those with stage IV breast cancer, &#62;36% of patients have lung metastasis17; however, the site-specific pattern and incidence of metastases can vary based on molecular subtype18,19,20,21. Patients with breast cancer-associated lung metastases have a median survival of only 21 months highlighting the need to identify effective treatments and novel biomarkers for this disease17. The use of experimental metastasis models, including the intravenous injection of tumor cells, will continue to advance our knowledge of this important clinical challenge. When combined with digital imaging pathology and the method of metastatic lung tumor burden analysis described within this protocol, tail-vein injections are a valuable tool for breast cancer metastasis research.

<table>
	<tbody>
		<tr>
			<td>alcohol prep pads</td>
			<td>Fisher Scientific</td>
			<td>22-363-750</td>
			<td>for cleaning tail prior to injection</td>
		</tr>
		<tr>
			<td>dissection scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-5</td>
			<td>for mouse dissection and lung tissue inflation</td>
		</tr>
		<tr>
			<td>DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>MT10013CV</td>
			<td>cell culture media base for MDA-MB-231 and MVT1 cell lines</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Salt Solution 1x</td>
			<td>Fisher Scientific</td>
			<td>MT21030CV</td>
			<td>used for resuspending tumor cells for injection</td>
		</tr>
		<tr>
			<td>ethanol (70 % solution)</td>
			<td>OSU</td>
			<td></td>
			<td>used to minimize mouse&#39;s fur during dissection; use caution - flammable</td>
		</tr>
		<tr>
			<td>Evan&#39;s blue dye</td>
			<td>Millipore Sigma</td>
			<td>E2129</td>
			<td>used at 1 % in sterile PBS for practice with tail-vein injection method; use caution - dangerous reagent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Millipore Sigma</td>
			<td>F4135</td>
			<td>cell culture media additive; used at 10% in DMEM</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fisher Scientific</td>
			<td>10-270</td>
			<td>for dissection and lung tissue inflation</td>
		</tr>
		<tr>
			<td>FVB/NJ mice</td>
			<td>The Jackson Laboratory</td>
			<td>001800</td>
			<td>syngeneic mouse strain for MVT1 cells</td>
		</tr>
		<tr>
			<td>hemacytometer (Bright-Line)</td>
			<td>Millipore Sigma</td>
			<td>Z359629</td>
			<td>for use in cell culture to obtain cell counts</td>
		</tr>
		<tr>
			<td>insulin syringe (28 G)</td>
			<td>Fisher Scientific</td>
			<td>14-829-1B</td>
			<td>for tail-vein injections (BD 329424)</td>
		</tr>
		<tr>
			<td>MDA-MB-231 cells</td>
			<td>ATCC</td>
			<td></td>
			<td>human breast cancer cell line</td>
		</tr>
		<tr>
			<td>MVT1 cells</td>
			<td></td>
			<td></td>
			<td>mouse mammary tumor cells</td>
		</tr>
		<tr>
			<td>needles (26 G)</td>
			<td>Fisher Scientific</td>
			<td>14-826-15</td>
			<td>used to inflate the mouse&#39;s lungs</td>
		</tr>
		<tr>
			<td>neutral buffered formalin (10%)</td>
			<td>Fisher Scientific</td>
			<td>245685</td>
			<td>used as a tissue fixative and to inflate lung tissue; use caution - dangerous reagent</td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice</td>
			<td>The Jackson Laboratory</td>
			<td>005557</td>
			<td>maintained by OSUCCC Target Validation Shared Resource</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin 100x</td>
			<td>ThermoFisher</td>
			<td>15140163</td>
			<td>cell culture media additive</td>
		</tr>
		<tr>
			<td>sterile gauze</td>
			<td>Fisher Scientific</td>
			<td>NC9379092</td>
			<td>for applying pressue to mouse&#39;s tail if bleeding occurs</td>
		</tr>
		<tr>
			<td>syringe (5 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-955-458</td>
			<td>used to inflate mouse lung tissue</td>
		</tr>
		<tr>
			<td>tail-vein restrainer</td>
			<td>Braintree Scientific, Inc.</td>
			<td>TV-150 STD</td>
			<td>used to restrain mouse for tail-vein injections</td>
		</tr>
		<tr>
			<td>Trypan blue (0.4 %)</td>
			<td>ThermoFisher</td>
			<td>15250061</td>
			<td>used in cell culture to assess viability</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25 %</td>
			<td>ThermoFisher</td>
			<td>25200-114</td>
			<td>used in cell culture to detach tumor cells from plate</td>
		</tr>
	</tbody>
</table>

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.

If using unlabeled cells for tail-vein injection, it may be difficult to confirm lung colonization until (1) the time of necropsy if macrometastases can be observed or (2) following histological analysis if microscopic metastases exist. With extensive metastatic lung tumor burden, mice will have labored breathing. As with any tumor study, mice should be carefully monitored throughout the study duration. The use of labeled cells is an easy way to confirm successful tail-vein injection; hence the use of luciferase-tagged M...

Watch this Scientific Journal Video about Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells at JoVE.com

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

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 ...

There is currently no gold standard for analyzing tail-vein injection induced metastatic lung tumor burdens. We utilized digital image analysis to accurately and reproducibly quantify lung metastases in a highly efficient manner. This method is time and cost effective.And, when coupled with digital image analysis, allows a highly accurate and comprehensive analysis of the resulting lung metastatic tumor burden. To prepare human breast cancer cells for injection, first, place an appropriate number of cells in a suitable cell culture medium, based on the number of mice and cell concentration to be used. When the cells reach an 80 to 90%confluency, wash the plates with PBS, and detach the cells in a minimal volume of trypsin, according to standard protocols.When the cells have detached, re-suspend the cells in fresh culture medium for counting, and collect the cells by centrifugation. Then, re-suspend the cells at the appropriate concentration in 100 microliters of PBS per mouse, on ice. Just before the injection, thoroughly re-suspend cells on ice to avoid clumping, and load 100 microliters of the cells into a 28-gauge insulin syringe.Keeping the syringe vertical, tap the shaft while slowly adjusting the plunger to remove any bubbles. If permitted, carefully recap the needle and bend the needle tip to a 20 to 30-degree angle. Restrain a greater than six-week-old female mouse in a plastic mouse restrainer and position the mouse on its side such that its lateral tail vein is easily viewed.Locate the ventral artery in line with the genitalia, a dorsal vein, and two lateral caudle veins, and use an aseptic wipe to clean the tail surface. Grasping the tail between the index finger and thumb of the non-dominant hand, apply slight tension, and beginning at the distal portion of the tail, insert the needle, bevel side up, parallel to the vein. Slowly deliver the entire volume of cells into the vein.A small volume of blood will likely be displaced after the injection. Apply gentle pressure with a sterile gauze to the injection site. Then discard the syringe in an appropriate sharps container, and place the mouse in a clean, ventilated cage with monitoring.Within no more than one hour of the tail-vein injection, perform live animal in vivo imaging to confirm a successful cell injection, and to obtain time zero data. To maintain the structural format of the lungs for histopathology, secure the mouse to a dissecting board and wet the fur with 70%ethanol. Open the thorax with a midline incision, extending the incision cranially and caudally through the peritoneum, and grasp the xiphoid process to allow removal of the diaphragm.Using a second pair of scissors, cut the ribs along each side of the sternum and carefully remove the ribcage to leave room for the lungs to expand. To isolate the trachea, remove the submandibular salivary glands and infrahyoid musculature, and place pins on either side of the trachea to prevent unwanted movement during the needle insertion. Load a three milliliter syringe, equipped with a 26-gauge needle, with two to three milliliters of 10%neutral buffered Formelin, and insert the needle into the trachea.Slowly inject the Formelin, while watching for the lungs to expand. Once the Formelin begins leaking out of the lungs, use forceps to pinch off the trachea and remove the needle. Then detach the entire respiratory apparatus and place the organs directly into a container of fresh Formelin.For analysis of the metastatic lung tumor burden, scan H&E stained lung tissue sections on a high resolution slide scanner at a 40 times magnification, and import the images into an appropriate image analysis software program. Adjust the parameters defining the shape and sparseness to best fit the images. To display the segmented areas of tumor metastases and normal lung tissue, using different colored labels for each tissue type, open the Decision Forest program, and follow the prompted series of yes or no questions to appropriately train each class for an image.To adjust the features for each class, apply filters to sharpen, blur, or sort by shape to enhance the accuracy of the algorithm. In this analysis, the tumor metastases appear in blue. The normal tissue is green, and the bronchiolar epithelium is yellow.Red blood cells can be observed in red, and air spaces appear in pink. When all of the features have been set, save the modified settings and apply the algorithm to the entire set or series of H&E stained tissues. Then export all of the output variables to allow the area in micron squared to be quantified for each tissue type, and to allow calculation of the percentages from the specimen total net tissue area.The presence of a bioluminescence signal within the thoracic space less than two hours after the tail-vein injection of luciferase-tagged human epithelial breast cancer cells can be considered confirmation of an accurate injection. In this experiment, the photon counts in the thoracic region increased over time and a strong bioluminescence signal was still present at day 24 post-injection. At the time of necropsy, many macroscopic lung lesions were observed in these mice.Using image analysis software and a custom algorithm as demonstrated, whole lung tissue can be segmented by different tissue features. By segmenting the lung tissue in this manner, the software can quantify various parameters. Here, raw data from an analysis performed on lung tissue from mice injected with human epithelial breast cancer cells followed by treatment with a drug designed to block metastatic colonization are shown.As demonstrated by these images, a difference in metastatic tumor burden between these treatment groups may have easily been overlooked, as the total number of lung nodules is no different. A comprehensive analysis of all the parameters, however, indicated a significant difference in the percent net lung metastasis area, underscoring the need for a thorough approach to metastatic lung tumor burden analysis A proper cell re-suspension without air bubbles is critical for preventing pulmonary embolisms, and a successful lung inflation helps to maintain the tissue structure integrity for an accurate image analysis. Immunostaining of the lung tissue can be performed to determine the presence of cells or proteins of interest within the lung lesions or the lung micro environment.

pathological-analysis-lung-metastasis-following-lateral-tail-vein

Research

JoVE Journal

Cancer Research

8.3K visualizações.  The Ohio State University Medical Center. There is currently no gold standard for analyzing tail-vein injection induced metastatic lung tumor burdens. We utilized digital image analysis to accurately and reproducibly quantify lung metastases in a highly efficient manner. This method is time and cost effective.And, when coupled with digital image analysis, allows a highly accurate and comprehensive analysis of the resulting lung metastatic tumor burden. To prepare human breast cancer cells for injection, first, place an appropr...