Name: intra-iliac artery injection for efficient and selective modeling of microscopic bone metastasis
Uploaded: 2016-09-26T12:30:00
Description: Watch this Scientific Journal Video about Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis at JoVE.com

Research in Zhang lab was supported by X. H.-F. Z.'s NCI CA151293, CA183878, Breast Cancer Research Foundation, U.S. Department of Defense DAMD W81XWH-13-1-0195, a Pilot Award of CA149196-04, McNair Medical Institute and by H.W.'s U.S. Department of Defense DAMD W81XWH-13-1-0296.

All animal work was done in accordance to the animal care guidelines of the Baylor College of Medicine.
1. Cell Preparation
Note: Different cancer cell lines can be used for IIA injection depending on research purposes. We have used breast cancer cell lines MCF7, 4T1, 4T07, MDA-MB-361, MDA-MB-231, MDA-MB-436 and prostate cancer cell line C4-2 in our research. We typically use both GFP- and firefly luciferase-labeled cancer cells for our study and show some data here f...

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Res. 10 (4), 494-503 (2012).</li><li>Gregory, L. S., Choi, W., Burke, L., Clements, J. A. <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+Cells+Induce+Osteolytic+Bone+Lesions+In+vivo+through+a+Reduction+in+Osteoblast+Activity+in+Mice.">Breast Cancer Cells Induce Osteolytic Bone Lesions In vivo through a Reduction in Osteoblast Activity in Mice.</a> PLoS ON. 8 (9), e68103 (2013).</li><li>Waning, D. L., 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=Excess+TGF-&#946;+mediates+muscle+weakness+associated+with+bone+metastases+in+mice.">Excess TGF-&#946; mediates muscle weakness associated with bone metastases in mice.</a> Nat Med. 21 (11), 1262-1271 (2015).</li><li>Simmons, J. 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L., 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=Tumor+microenvironment+regulates+metastasis+and+metastasis+genes+of+mouse+MMTV-PymT+mammary+cancer+cells+in+vivo.">Tumor microenvironment regulates metastasis and metastasis genes of mouse MMTV-PymT mammary cancer cells in vivo.</a> Vet Pathol. 51 (4), 868-881 (2014).</li><li>Xiang, 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=CXCR4+Protein+Epitope+Mimetic+Antagonist,+POL5551,+Disrupts+Metastasis+and+Enhances+Chemotherapy+Effect+in+Triple+Negative+Breast+Cancer.">CXCR4 Protein Epitope Mimetic Antagonist, POL5551, Disrupts Metastasis and Enhances Chemotherapy Effect in Triple Negative Breast Cancer.</a> Mol Cancer Ther. 14 (11), 2473-2485 (2015).</li><li>Wang, 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=The+osteogenic+niche+promotes+early-stage+bone+colonization+of+disseminated+breast+cancer+cells.">The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells.</a> Cancer Cell. 27 (2), 193-210 (2015).</li><li>Hoffmann, 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=Characterization+of+new+estrogen+receptor+destabilizing+compounds:+effects+on+estrogen-sensitive+and+tamoxifen-resistant+breast+cancer.">Characterization of new estrogen receptor destabilizing compounds: effects on estrogen-sensitive and tamoxifen-resistant breast cancer.</a> J Natl Cancer Inst. 96 (3), 210-218 (2004).</li><li>Tannehill-Gregg, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroPET/SPECT/CT+imaging+of+small+animal+models+of+disease.">MicroPET/SPECT/CT imaging of small animal models of disease.</a> Am J Pathol. 182 (2), 319-324 (2013).</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=Canine+prostate+cancer+cell+line+(Probasco)+produces+osteoblastic+metastases+in+vivo.">Canine prostate cancer cell line (Probasco) produces osteoblastic metastases in vivo.</a> Prostate. 74 (13), 1251-1265 (2014).</li><li>Amend, S. 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Although only the iliac artery is the target of injection for cancer cells, we recommend the separation of both iliac vein and artery from surrounding tissues, and to lift them together as a bundle. This is because the vein and artery extensively contact with each other, and the venous vessel wall is thin and is easy to break. Therefore, for a successful injection, it saves time and effort to hold up the two vessels together, although cancer cells are injected only to the artery. A 4-0 silk suture is used to help this pr...

Metastases account for over 90% of deaths caused by solid tumors. Bone is the most common organ affected by metastases of various cancer types, especially breast and prostate cancers. When diagnosed in the clinic, bone metastases usually have already entered advanced stages with either osteolytic or osteoblastic alterations in bone, often accompanied with neurological symptoms.
Previous studies predominantly focused on the overt osteolytic bone metastases1-3, however we currently have limited understanding of micrometastases in bones before the onset of the osteolytic process. This is at least partly due to lack of appropriate experimental models and approaches. Genetically engineered mouse models of breast cancer often metastasize to lungs, but much less efficiently to bones4. Likewise, the orthotopically transplanted tumors rarely develop spontaneous bone metastases, with some bone-tropical 4T1 mammary carcinoma sub-clones and MSP overexpressed PyMT transgenic mouse model as exceptions5-7. Intra-tibial drilling can deliver cancer cells to the bone8-10, but it also incurs damage and inflammation to local tissues. Currently intra-cardiac injection of breast cancer cell lines has been the major approach to investigate bone colonization11-13. However, after cancer cells are introduced into left ventricle only a limited proportion will finally reach bone and bone marrow, making it difficult to track microscopic metastases in a quantifiable fashion.
In this study, we establish a technique, namely intra-iliac artery (IIA) injection14, to selectively deliver cancer cells into hind limb tissues, thereby enriching cancer cells in bone and bone marrow without causing damage to local tissues. Because of the bone specificity, this approach also allows enough time for indolent cancer cells to eventually colonize before the animals succumb to primary tumors or metastases in other vital organs. When combined with a variety of other techniques, such as bioluminescence imaging, immunofluorescence staining and bone histomorphometry, IIA injection is potentially useful for a wide scope of research purposes related to bone metastases, especially to track the progression from single cancer cells to multi-cell micrometastases. In particular, we demonstrated that IIA injection enables us to visualize the interactions between cancer cells and various types of surrounding cells in the bone microenvironment.

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			<td height="21" style="height:21px;width:216px;">Materials</td>
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			<td style="width:203px;"></td>
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			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:303px;">HyClone</td>
			<td style="width:123px;">SH30022.01</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:303px;">Gibco</td>
			<td style="width:123px;">16000</td>
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			<td height="21" style="height:21px;width:216px;">Pen/Strep Amphatericin B</td>
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			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:303px;">Lonza Biowhittaker</td>
			<td style="width:123px;">17-516F</td>
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			<td height="21" style="height:21px;width:216px;">Trypsin/EDTA solution</td>
			<td style="width:303px;">HyClone</td>
			<td style="width:123px;">SH30042.01</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">45uM cell strainer</td>
			<td style="width:303px;">VWR International Laboratory</td>
			<td style="width:123px;">195-2545</td>
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			<td height="21" style="height:21px;">MediGel CPF with carprofen&#160;</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td>For pain management</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine&#160;</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
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			<td>For pain management</td>
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			<td height="21" style="height:21px;width:216px;">Estradiol pellet</td>
			<td style="width:303px;">Innovative Research of America</td>
			<td style="width:123px;">SE-121</td>
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			<td height="21" style="height:21px;width:216px;">Ketamine and xylazine</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
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			<td height="21" style="height:21px;width:216px;">Vet ointment</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
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			<td>Avoid eye dryness</td>
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			<td height="21" style="height:21px;width:216px;">Shaver</td>
			<td style="width:303px;">Oster</td>
			<td style="width:123px;">78005-050</td>
			<td>For furred mice</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropyl ethanol</td>
			<td style="width:303px;">ACROS</td>
			<td style="width:123px;">67-63-0</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Betadine surgical scrub</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#10 scalpel blades</td>
			<td style="width:303px;">Ted Pella, Inc</td>
			<td style="width:123px;">549-3CS-10</td>
			<td>Multiple</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">No. 3 handle</td>
			<td style="width:303px;">Ted Pella, Inc</td>
			<td style="width:123px;">541-31</td>
			<td>Need to be autoclaved</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile surgical drape</td>
			<td style="width:303px;">Sai Infusion Technology</td>
			<td style="width:123px;">PSS-SD1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight forceps&#160;</td>
			<td style="width:303px;">Roboz Surgical Instrument</td>
			<td style="width:123px;">RS-5132</td>
			<td>Need to be autoclaved</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight fine forceps</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">11253-20</td>
			<td>Need to be autoclaved</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Edged fine forceps</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">11253-25</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 Vicryl silk suture</td>
			<td style="width:303px;">Johnson &#38; Johnson Health Care</td>
			<td style="width:123px;">J214H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">31G insuline syringes</td>
			<td style="width:303px;">BD</td>
			<td style="width:123px;">328418</td>
			<td>Multiple</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Q-tips cotton swabs (Sterile)</td>
			<td style="width:303px;">VWR International Laboratory</td>
			<td style="width:123px;">89031-272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Skin glue</td>
			<td style="width:303px;">Henry Schein Animal Health</td>
			<td style="width:123px;">31477</td>
			<td>For surgery site skin closure</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ear Tag Applicator</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">24220-00</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ear tags</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">24220-50</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">D-luciferin</td>
			<td style="width:303px;">Gold Biotechnology</td>
			<td style="width:123px;">LUCK</td>
			<td>Avoid light and put on ice</td>
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			<td height="21" style="height:21px;width:216px;">28G insulin syringes</td>
			<td style="width:303px;">BD</td>
			<td style="width:123px;">329410</td>
			<td>For intra-orbital injection</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformadehyde</td>
			<td style="width:303px;">Alfa Aesar</td>
			<td style="width:123px;">30525-89-4</td>
			<td>For tissue fixation</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:303px;">OmniPur</td>
			<td style="width:123px;">4050</td>
			<td>For bone tissue decalficication</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:303px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissection microscope</td>
			<td style="width:303px;">Leica</td>
			<td style="width:123px;">Leica S6E stereo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS Lumina II imaging system</td>
			<td>Advanced Molecular Vision</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibodies</td>
			<td></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-GFP antibodies (JL-8)</td>
			<td>Clontech</td>
			<td>632381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-ALP antibodies</td>
			<td>Abcam</td>
			<td>ab108337</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Osterix antibodies</td>
			<td>Abcam</td>
			<td>ab22552</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 illustrates the anatomical location and relationship of common iliac artery (red) and vein (blue).
Figure 2 shows relative position of iliac vessels and nerves under dissection microscopy. As depicted in Figure 2A, the vessels and nerves are right beneath the peritoneal wall and can be revealed after the skin incision is made and the peritoneum is pushed away. The commo...

Watch this Scientific Journal Video about Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis at JoVE.com

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

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 ...

The overall goal of this procedure is to establish experimental bone metastasis by delivering cancer cells directly to hind-limb tissues, including the bones, via intra-iliac artery injection. So this method can help us answer the key questions about breast cancer bone colonization, especially in the early phase of this process, how breast cancer cells interact with microenvironment cells in the bone. We did iliac artery injection today.The main advantage of this technique is that it efficiently and selectively establish an experimental microscopic bone metastatis model for various cancer types without causing local tissue damage. Generally, individuals new to this method will struggle because finding an injection that's accurate, iliac artery injection position on the microscope may take practice to achieve higher than ninety percent injection success rate. Begin by confirming the appropriate level of sedation by toe pinch of a five to six week old twenty gram mouse.After applying eye ointment, wipe the shaved right groin area several times with sequential, sterile, 70%isopropyl ethanol-soaked cotton swabs, followed by a betadine surgical scrub. Next, cover all of the mouse with a sterile drape. Then, use a sterile number ten carbon steel scalpel blade and a number three handle to make a one to 1.2 centimeter skin incision between the fourth and fifth nipples in the lower right abdomen.And transfer the animal under a standard benchtop dissection microscope. Under the 4X objective, insert blunt separation forceps into the incision between the fatty tissue and the peritoneum, and push the tissues outwards on both sides to reveal the iliac vessels and nerves. Next, use straight, fine forceps to break through the connective tissue between the vessels and the nerve closest to the vessels.Then, slide angled forceps through the connective tissue and beneath the iliac vessels, while inserting the straight forceps into the connective tissues on the other side of the vessels to guide the angled forceps through the tissue. We recommend separating and lifting the ilial rim and artery as a bundle for this step. Although the tumor cells are injected only into the artery, it saves time and effort to isolate both vessels together.Once the angled forceps are in place, further separate the iliac vein and artery from the surrounding tissue, keeping the vessels on top of the angled forceps during the dissection. When the vessels are clear, use the straight forceps to deliver the tip of a 4-0 silk suture to the angled forceps and pull the suture slowly and gently beneath the vessels. Both of the iliac common vein and artery vessels should then be held up by the suture.When the vessels are ready, pipette the tumor cells up and down several times to re-suspend them into a single cell solution and to break up any clumps. Then, aspirate 100 microliters of cells per mouse into a 31 gauge insulin syringe. Now, slide the angled forceps back under the vessels using the suture as a guide, and open the forceps so that the vessels rest gently on the tips.With the other hand, insert the needle tip bevel-side up into the lumen of the section of the artery between the forceps tips. Depress the plunger slowly to inject the cells. The cell suspension will flush the blood through the vessel.When all of the cells have been delivered, gently remove the needle, keeping the vessels raised on the forceps to prevent bleeding. Then, pull away the suture. Finally, quickly remove the forceps and press a cotton swab onto the incision for five to 10 minutes to stop the bleeding.Here, a representative whole-animal bioluminescent image after intra-iliac artery injection of five times 10 to the fifth GFP luciferin-labeled MCF-7 breast cancer cells followed by d-luciferin administration by retro-orbital sinus injection is shown. At day 14 post injection, a strong bioluminescence signal was obtained from the intra-iliac artery injected hind bone, but not from the contralateral control bone, confirming the specific localization of the injected cells. Sectioning an H&E staining of the tumor-bearing bones also demonstrated a compact, cobblestone-like cell population with large nuclei indicative of microscopic MCF-7 metastatic lesions in the bone tissue 14 days after intra-iliac artery injection.Further, immunoflourescent staining of the bone sections also revealed a co-mingling of the MCF-7 cells with osteoblasts and preosteoblasts. Once mastered, this technique can be completed in ten to fifteen minutes. Following these procedures, you can use other methods to answer additional questions.For instance, we can use RNA-Seq to profile the microenvironment cells in the metastasis niche and compare them to normal cells. This technique provides one way for breast cancer researchers and prostate cancer researchers to study bone micrometastasis in animals. After watching this video, you should have a good understanding of how to establish a breast cancer bone metastasis model by intra-iliac artery injection.Please take appropriate precautions because working with ketamine and xylazine could be extremely hazardous.

intra-iliac-artery-injection-for-efficient-selective-modeling

Research

JoVE Journal

Cancer Research

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. ...

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 ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

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 Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

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

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

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

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

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...