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 Video 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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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=The+macrophage-stimulating+protein+pathway+promotes+metastasis+in+a+mouse+model+for+breast+cancer+and+predicts+poor+prognosis+in+humans.">The macrophage-stimulating protein pathway promotes metastasis in a mouse model for breast cancer and predicts poor prognosis in humans.</a> Proc Natl Acad Sci U S A. 104 (18), 7570-7575 (2007).</li><li>Li, X., 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=Loss+of+TGF-beta+Responsiveness+in+Prostate+Stromal+Cells+Alters+Chemokine+Levels+and+Facilitates+the+Development+of+Mixed+Osteoblastic/Osteolytic+Bone+Lessions.">Loss of TGF-beta Responsiveness in Prostate Stromal Cells Alters Chemokine Levels and Facilitates the Development of Mixed Osteoblastic/Osteolytic Bone Lessions.</a> Mol. Cancer. 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. H., Levine, A. L., Nadella, M. V., Iguchi, H., Rosol, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+zoledronic+acid+and+osteoprotegerin+on+growth+of+human+lung+cancer+in+the+tibias+of+nude+mice.">The effect of zoledronic acid and osteoprotegerin on growth of human lung cancer in the tibias of nude mice.</a> Clin Exp Metastasis. 23 (1), 19-31 (2006).</li><li>Slyfield, C. R., Tkachenko, E. V., Wilson, D. L., Hernandez, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Dynamic+Bone+Histomorphometry.">Three-Dimensional Dynamic Bone Histomorphometry.</a> J Bone Miner Res. 27 (2), 486-495 (2012).</li><li>Koba, W., Jelicks, L. A., Fine, E. 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>
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		<tr height="21">
			<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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		<tr height="21">
			<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>
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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>
		</tr>
		<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>
		</tr>
		<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 Video at JoVE.com

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis Video (Video) | JoVE

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 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 ...

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A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer Video (Video) | JoVE

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

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis Video (Video) | JoVE

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

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland Video (Video) | JoVE

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

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment Video (Video) | JoVE

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

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

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

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells Video (Video) | JoVE

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

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging Video (Video) | JoVE

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

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone Video (Video) | JoVE

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

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells Video (Video) | JoVE

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

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma Video (Video) | JoVE

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