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.
转移占引起实体瘤死亡的90%以上。骨是受各种癌症类型,特别是乳腺癌和前列腺癌的转移中最常见的器官。当诊断在临床上,骨转移一般都已经进入高级阶段,在骨无论是溶骨性或成骨细胞的改变,常伴有神经症状。
以往的研究主要集中在明显溶骨骨转移1-3,但是我们目前有溶骨过程来临前限制骨骼微的理解。这至少部分是由于缺乏合适的实验模型和方法。乳腺癌的基因工程小鼠模型常转移至肺部,但更高效地骨4。同样,原位移植瘤很少自发的骨转移,一些骨热带4T1乳腺carcinom子的克隆和MSP过表达PyMT转基因小鼠模型例外5-7。帧内胫钻孔可以提供癌细胞的骨8-10,但它也导致损伤和炎症到局部组织。目前的乳腺癌细胞系心内注射已经调查骨定植11-13的主要方法。然而,在癌症细胞导入左心室只有有限的比例将最终达到骨和骨髓,使得它难以跟踪在一个量化的方式微观转移。
在这项研究中,我们建立了一种技术,即帧内髂动脉(ⅡA)注射14,癌细胞选择性地递送到后肢组织中,从而丰富癌细胞在骨和骨髓而不导致破坏局部组织。由于骨特异性,该方法还允许有足够的时间好逸恶劳癌细胞的一个前殖民最终imals屈从于原发肿瘤或转移的其他重要器官。当与各种其他技术,如生物发光成像,免疫荧光染色和骨形态计量学结合,ⅡA注射是相关的骨转移研究目的宽范围可能有用的,尤其是跟踪从单个癌细胞多小区进展微转移。特别是,我们表明,IIA族注射使我们能够可视化癌细胞和各种类型的在骨微环境周围细胞之间的相互作用。
虽然只有髂动脉被注射癌细胞的目标,我们建议既髂静脉和动脉从周围组织中分离,并把它们一起抬起作为包。这是因为静脉和动脉广泛相互接触,与静脉血管壁薄,容易断裂。因此,对于一个成功的注射,这样可以节省时间和精力,将两个容器保持在一起,虽然癌细胞仅注射到动脉。 4-0丝线来帮助这个过程如图2所示,缝合线还可以帮助它应该出现止血。
需要?…
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 |