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

Summary

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.

Abstract

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.

Introduction

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 metastases^1-3, 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 exceptions^5-7. Intra-tibial drilling can deliver cancer cells to the bone^8-10, 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 colonization^11-13. 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.

Protocol

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.

1. Maintain cells in DMEM containing 10% FBS and 0.1 mg/ml penicillin-streptomycin at 37 °C in 5% CO₂.
2. Before IIA injection, trypsinize cells at 80 - 90% confluence with 0.25% Trypsin/EDTA solution. Use cold PBS to wash cells twice, and re-suspend cells in 10 ml PBS for cell counting. To remove PBS, centrifuge cells at 800 x g for 5 min.
3. Re-suspend cells at a concentration of 5 x 10⁶ cells/ml in ice-cold PBS.
4. Use 100 µl GFP- and firefly luciferase-labeled cancer cells for IIA injection of one mouse.
   Note: Therefore, the number of cells received by each animal is 5 x 10⁵. This number may need to be altered based upon the aggressiveness of cell lines.
5. Keep cell suspensions on ice.
   Note: Vibrations may be needed to prevent cell aggregation every few minutes until it is ready for injection. For some cell lines (such as MDA-MB-231), more stringent procedures (e.g., passing through 45 µm cell strainer) may be needed to prevent aggregation. Please note that the clog of vessels may cause tissue necrosis, and therefore confounding the experiments.

2. Animal Preparation

1. For animal pain management, give 5 mg/kg/day carprofen (or other analgesic) with dietary supplement to the mice no less than 24 hr before surgery. Administer a dose of 0.1 mg/ml buprenorphine subcutaneously 60 min prior to the surgery. Note: an optional procedure is to implant estradiol pallet into the back of animals to provide extra estradiol. This will accelerate the grow of ER+ cancer cells (e.g., MCF-7 cells)⁹.
2. Anesthetize a 4 - 6 week old mouse (approximately 20 g) with ketamine (80 - 100 mg/kg) and xylazine (10 -12.5 mg/ml) by intraperitoneal injection.
3. Confirm the appropriate level of sedation of a 20 g mouse by toe pinch and the observation of a 50% reduction in respiratory rate and pink mucous membranes (e.g., mouse ear skin and paw skin). Keep monitoring these vital signs every 15 min during the surgery. Note: No movement of the animal indicates that the animal is sufficiently anesthetized and ready for surgery.
4. Use vet ointment on eyes to prevent dryness.
5. Shave the mouse on the lower abdomen, especially the right groin area where the surgery and injection will take place.
   Note: This is not necessary if athymic nude mice are used.
6. Put the mouse on its back, spread the legs in their natural positions and stick the toes to mobile dissection cardboard with tape.
7. Wipe the right groin area with 70% isopropyl ethanol soaked sterile cotton swabs, followed with betadine surgical scrub several times.

3. The Common Iliac Vein and Artery Location and Separation

1. Make a skin incision of 1.0 - 1.2 cm long between the 4^th and 5^th nipples in the lower right abdomen using sterile, #10 carbon steel scalpel blades held in a No. 3 handle. Then use a sterile surgical drape to cover the animal body except the incision site.
2. Move the animal to standard bench top dissection microscope. Use a magnification of 4X for injection.
3. Under 4X magnification of the dissection microscope, insert blunt separation forceps between the fatty tissue and the peritoneum, and push the tissues outwards on both sides to expose the iliac vessels and nerves (see Figure 1).
   Note: The artery between aorta from the midline and the lower part of artery branches is called common iliac artery. This is where the injection takes place.
4. Use one pair of straight fine forceps to break through the connective tissue (like a membrane) between vessels and the nerve, closer to the vessels.
5. Switch the straight fine forceps to the left hand. Grab a pair of angled fine forceps using the right hand. Insert the right hand forceps into same position where connective tissues have been broken. And then stick the right hand forceps through, beneath the vessels.
6. At the same time, insert the left hand forceps into the connective tissues on the left side of the vessels to guide the right hand forceps going through.
7. Once the right hand forceps gets beneath the vessels, try to separate the vessels from the surrounding tissue a little bit more, and then keep them on top of the right hand forceps.
8. Use left hand forceps to deliver the tip of a 4-0 silk suture to the right hand forceps, and then pull the suture gently and slowly through beneath the vessels. Note: Now both common vein and artery vessels are lifted up by the suture (see Figure 2).

4. Injection and Post-injection Care

1. Prepare the cells for injection in a 31 G insulin syringe. Use 100 µl of cell suspension in PBS per injection. Make sure to pipette up and down several times to separate the cells and avoid aggregation so that the injected cells will not clog and block the blood flow.
2. With the angled forceps in the left hand, put the forceps beneath the vessels along the guidance of the suture, and then open the two arms of the forceps, having the vessels held gently on the forceps.
   Note: The interval between the two arms of the forceps will be the injection site.
3. Hold the 31 G needle with 100 µl cell suspensions in the right hand, with the bevel of the needle upward, ready for injection.
4. Insert the needle into the artery lumen (blood will enter the needle tip). Then push cell suspension slowly in to inject the cells. The cell suspension will push away the red blood in the vessel. Try not to break the vessels or leak the cell suspension out.
5. When the injection is finished, gently pull back the needle and keep holding the vessels up on the left hand forceps to stop bleeding. Then pull away the suture.
6. Pull away the left hand forceps, and quickly use a cotton swab to press the artery incision area to stop bleeding.
   Note: Usually 5 - 10 min continuous pressure is sufficient to stop bleeding.
7. When bleeding stops, use skin glue to close the skin edges of the surgery area. Make an ear-tag, and check for bioluminescence signaling to examine the distribution of injected cells.
   Note: A successful injection will result in an image similar to Figure 3.
   1. Check bioluminescence signaling by injection of 100 µl 15 mg/ml luciferin in PBS at 75 mg/kg mouse concentration via the intra-orbital sinus.
      1. For intra-orbital injection, put fingers on both sides of the mouse eye, use pressure to make eye ball slightly pop out from the eye frame, insert 28 G insulin syringe needles between eye ball and nose, and then slowly push syringe to inject luciferin.
         Note: At the correct position, the needle should be able to reach a depth of 2 - 3 mm before hitting hard tissue (bone).
      2. Place the mouse into imaging system for in vivo whole animal imaging for 10 - 60 sec within 5 min (see Figure 3).
         Note: An optional procedure is to implant estradiol pellet into the back of animals to provide extra estradiol. This will accelerate the growth of ER+ cancer cells (e.g., MCF-7 cells)¹⁵.
8. Leave the mouse on a warm heating pad until it wakes up. Monitor the bleeding, swelling, signs of dehiscence and pain during the post-surgical period. Put the mice back to the cage with analgesic coverage (carprofin dietary supplement suggested) given for pain management until no signs of pain. Pay close attention and care to the animals during 7 days following surgery.

5. Monitoring Metastatic Growth

1. Histomorphometry:
   1. Harvest tumor bearing bone tissues, fix the bones in 4% paraformaldehyde for 24 hr, then decalcify them in pH 7.0 14% EDTA solution for 3 - 5 days, and subject them to paraffin embedding as previously described¹⁴.
   2. Section the paraffin-embed bone tissues at the thickness of 3 µm and perform standard histological method of Hematoxylin/Eosin staining as previously described¹⁴.
2. Immunohistochemistry:
   1. Subject paraffin-embedded tumor bearing bone slides to immunohistochemistry staining for various purposes as previously described¹⁴. Briefly, immunostain MCF7 cancer cells by anti-GFP antibodies, and immunostain osteogenic cells (pre-osteoblasts and osteoblasts) by anti-Osterix antibodies and anti-Alkaline phosphatase (ALP) antibodies, respectively.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
45 μM 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
31 G 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
28 G 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