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 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.
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.
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 from the GFP-Luciferase-labeled MCF7 cell line.
2. Animal Preparation
3. The Common Iliac Vein and Artery Location and Separation
4. Injection and Post-injection Care
5. Monitoring Metastatic Growth
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 common iliac vein is on the left, and is bigger and darker compared to the artery. The artery is in the middle, and looks pink. It is thinner than the vein but has thicker muscle wall. The vein and artery are parallel and closely connected with each other. Farther on the right is the white lumbosacral nerve. Figure 2B shows common iliac vessels separated from the surrounding connective tissue, muscles and nerves, and lifted up by a 4-0 silk suture. The common iliac vein, artery, and lumbosacral nerve are also indicated.
Figure 3 shows representative in vivo and ex vivo bioluminescence images of animals after intra-iliac artery injection. Five x 105 GFP-luciferin-labeled MCF7 cells in 100 µl were administrated to the right hind limb of the mouse by intra-iliac artery injection. D-luciferin was then administrated by intra-orbit sinus injection, followed by the in vivo whole animal bioluminescence imaging. The injected MCF7 cells were enriched at the right hind limb of the mouse as indicated by the bioluminescence signals (Figure 3A). The in vivo bioluminescence signals of the whole animal were tracked every 3 days or every week, and then the mouse bone tissues were harvested at day 14 post-injection when the whole animal bioluminescence signal reached a certain threshold (Photon flux >104). After D-luciferin administration, the bone tissues from intra-iliac artery injected mouse were quickly harvested and immersed in PBS for ex vivo imaging. The strong bioluminescence signal from the intra-iliac artery injected right hind bone but not from the left control bone showed the specific localization of injected cells (Figure 3B).
Figure 4 shows representative images of histological and immunofluorescent staining. When the tumor bearing bones were harvested, they were subjected to paraformaldehyde fixation, EDTA decalcification, and then paraffin-embedding. Three µm bone slides were prepared and standard H&E staining were performed. The compact cobblestone-like cells with larger nuclei were microscopic MCF7 metastatic lesions in the bone tissue 14 days after intra-iliac artery injection, as indicated by the red arrows. Bone marrow (BM) and the large pink flat trabecular bones (TB) with sparse nuclei are so labeled (Figure 4A). Figure 4B shows GFP-labeled MCF7 cells (green), ALP-labeled osteoblasts (red in the left image), and Osterix-labeled pre-osteoblasts (red in the right image) after immunofluorescent staining. Blue DAPI staining indicates the nucleus.
Figure 1: Anatomy of common iliac artery (red) and vein (blue) in mouse. Abdominal aorta, common iliac artery, external iliac artery, and femoral artery are shown as indicated. Injection is performed at common iliac artery from aorta toward femoral artery direction. Please click here to view a larger version of this figure.
Figure 2: Iliac vessels and nerves under standard dissection microscope with 4X magnification. (a) Image of the intact vessels and nerves. (b) Image of lifted vessels. Please click here to view a larger version of this figure.
Figure 3: Representative in vivo and ex vivo bioluminescence images of mouse after intra-iliac artery injection. (a) The in vivo bioluminescence signal of the whole animal right after injection. (b) The ex vivo bioluminescence signal of left control bone and the right injected bone from injected animals that were harvested 14 days later. All the bioluminescence signals were measured by following the manufacturer's recommended procedure and settings. Please click here to view a larger version of this figure.
Figure 4: Representative images of histological and immunofluorescent staining. (a) Representative H&E staining image of MCF7 tumor bearing bone tissue after intra-iliac artery injection. Scale bar = 25 µm. Note: HE staining is not the effective way to detect tumors. In lower magnification, HE staining is not sensitive enough to distinguish tumors. In higher magnification, it is not efficient to scan all the areas. (b) Immunofluorescent staining images of the MCF7 tumor bearing bone tissue. Green: GFP-labeled MCF7 cells, Red in the left image: ALP-labeled osteoblasts, Red in the right image: Osterix-labeled pre-osteoblasts. Blue DAPI staining indicates the nucleus. Scale bar = 25 µm. Please click here to view a larger version of this figure.
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 process as shown in Figure 2. The suture may also help stop bleeding should it occur.
Most steps of IIA injection need to be performed under the dissection microscope. The mouse vessels are soft and small, which makes the procedures challenging. However, after sufficient practice, the success rate can reach 90 – 100% in our experience.
With this technique, researchers may be able to establish bone colonization models of their favorite cancer cell models, including those traditionally thought "non-metastatic". MCF-7 cells represent such an example. Indeed, when arriving in the bone microenvironment, MCF-7 cells undergo a short dormancy before taking off to colonize. Very few spontaneous bone metastases have been detected in animals carrying orthotopic MCF-7 xenografts. However, failure may well result from the residual quantity of disseminated tumor cells, the slow initiation of colonization, and the more aggressive growth of orthotopic tumors (that kills animals before bone lesions can establish). Thus, lack of detection cannot be taken as evidence against MCF-7 cells' ability to metastasize. In fact, indolent or even dormant bone micrometastases may be more prevalent in human breast cancer patients, as suggested by years-to-decades dormancy that is often seen in the clinic.
IIA injection can be applied not only to luminal or basal breast cancer cells, but also to other cancer types such as prostate cancer. When combined with different choices of techniques monitoring bone disease, intra-iliac artery injection can make a significant contribution to many research purposes. We commonly use bioluminescence signaling to trace bone metastasis progression after intra-iliac artery injection, and then harvest tumor-bearing bones for fluorescent immunohistochemistry staining to define interactions between cancer cells and surrounding bone microenvironment niches. Bone histomorphometry16-17 and µCT18-20 can be used to evaluate bone structure alterations and other anatomical details of bone that are caused by the inoculation of cancer cells. Proper considerations and combinations should be determined by each group for their specific research purpose.
Similar to intra-tibial8-10 and intra-cardiac inoculation11-13, a caveat of intra-iliac artery injection is that it does not recapitulate early steps in metastatic process prior to embolism and entry of tumor cells into the circulation. Ideally, a spontaneous metastasis process starting from orthotopic tumors would be needed to fully recapitulate the metastasis cascade. However, this process is highly inefficient in most models, with only a few exceptions such as some bone-tropic 4T1 sub-clones5-6 and MSP overexpressed PyMT transgenic mouse model7. We hope the IIA injection will provide novel insights into the bone colonization process, which can in turn facilitate the design and development of truly efficient spontaneous bone metastasis models for pre-clinical studies.
The authors have nothing to disclose.
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.
Materials | |||
DMEM | HyClone | SH30022.01 | |
FBS | Gibco | 16000 | |
Pen/Strep Amphatericin B | Lonza Biowhittaker | 17-745E | |
PBS | Lonza Biowhittaker | 17-516F | |
Trypsin/EDTA solution | HyClone | SH30042.01 | |
45uM cell strainer | VWR International Laboratory | 195-2545 | |
MediGel CPF with carprofen | Controlled item from veterinary care in BCM | For pain management | |
Buprenorphine | Controlled item from veterinary care in BCM | For pain management | |
Estradiol pellet | Innovative Research of America | SE-121 | |
Ketamine and xylazine | Controlled item from veterinary care in BCM | ||
Vet ointment | Controlled item from veterinary care in BCM | Avoid eye dryness | |
Shaver | Oster | 78005-050 | For furred mice |
Isopropyl ethanol | ACROS | 67-63-0 | |
Betadine surgical scrub | Controlled item from veterinary care in BCM | ||
#10 scalpel blades | Ted Pella, Inc | 549-3CS-10 | Multiple |
No. 3 handle | Ted Pella, Inc | 541-31 | Need to be autoclaved |
Sterile surgical drape | Sai Infusion Technology | PSS-SD1 | |
Straight forceps | Roboz Surgical Instrument | RS-5132 | Need to be autoclaved |
Straight fine forceps | Fine Science Tools | 11253-20 | Need to be autoclaved |
Edged fine forceps | Fine Science Tools | 11253-25 | Need to be autoclaved |
4-0 Vicryl silk suture | Johnson & Johnson Health Care | J214H | |
31G insuline syringes | BD | 328418 | Multiple |
Q-tips cotton swabs (Sterile) | VWR International Laboratory | 89031-272 | |
Skin glue | Henry Schein Animal Health | 31477 | For surgery site skin closure |
Ear Tag Applicator | Fine Science Tools | 24220-00 | |
Ear tags | Fine Science Tools | 24220-50 | |
D-luciferin | Gold Biotechnology | LUCK | Avoid light and put on ice |
28G insulin syringes | BD | 329410 | For intra-orbital injection |
Paraformadehyde | Alfa Aesar | 30525-89-4 | For tissue fixation |
EDTA | OmniPur | 4050 | For bone tissue decalficication |
Name | Company | Catalog Number | Comments |
Equipment | |||
Dissection microscope | Leica | Leica S6E stereo | |
IVIS Lumina II imaging system | Advanced Molecular Vision | ||
Name | Company | Catalog Number | Comments |
Antibodies | |||
Anti-GFP antibodies (JL-8) | Clontech | 632381 | |
Anti-ALP antibodies | Abcam | ab108337 | |
Anti-Osterix antibodies | Abcam | ab22552 |