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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: September 26th, 2016

DOI:

10.3791/53982

1Lester and Sue Smith Breast Center, Baylor College of Medicine, 2Department of Molecular and Cellular Biology, Baylor College of Medicine, 3Graduate Program in Developmental Biology, Baylor College of Medicine, 4Department of Molecular and Human Genetics, Baylor College of Medicine, 5McNair Medical Institute, Baylor College of Medicine, 6Dan L. Duncan Cancer Center, Baylor College of Medicine

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 ex....

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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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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.......

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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.......

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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.

....

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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
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

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