顕微鏡骨転移の効率的かつ選択的モデリングのためのイントラ腸骨動脈注入
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
転移は、固形腫瘍による死亡の90％以上を占めています。骨は、様々な癌の種類、特に乳癌および前立腺癌の転移によって影響を受ける最も一般的な臓器です。クリニックで診断すると、骨転移は通常、すでに多くの場合、神経学的症状を伴う、骨に溶骨性または骨芽細胞の変化のいずれかで進行した段階に入ってきました。
主に明白な溶骨性骨転移1-3に焦点を当てた以前の研究は、しかし、我々は現在、溶骨性プロセスの開始前に、骨の中に微小転移の理解が限られています。これは、少なくとも部分的には、適切な実験モデルとアプローチの欠如にあります。乳癌の遺伝子操作されたマウスモデルは、多くの場合、肺に転移するが、はるかに少ない効率的に骨4へ。同様に、同所移植された腫瘍はほとんどいくつかの骨の熱帯4T1乳腺carcinomで、自発的な骨転移を発症しませんサブクローンおよびMSPは、例外5-7としてPyMTトランスジェニックマウスモデルを過剰発現しました。イントラ脛骨掘削は骨8-10に癌細胞を提供することができますが、それはまた、局所組織への損傷および炎症を招きます。現在の乳癌細胞株の心臓内注射は、骨定着11-13を調査するための主要なアプローチとなっています。癌細胞は、左心室の中に導入された後、しかし、限られた割合は、最終的にそれが困難な定量化方法で微小転移を追跡すること、骨および骨髄に到達します。
本研究では、選択的に局所組織への損傷を引き起こすことなく、骨及び骨髄中の癌細胞を濃縮、後肢組織に癌細胞を送達するために、技術、すなわち内腸骨動脈（IIA）注入14を確立します 。なぜなら骨特異性のため、このアプローチは、無痛性癌細胞は最終的に、ANの前にコロニーを形成するのに十分な時間を可能にしますimalsは、他の重要な臓器における原発腫瘍または転移に屈します。このような生物発光イメージング、免疫蛍光染色および骨組織形態計測などの他の種々の技術と組み合わせると、IIA注入は、特にマルチセルに単一の癌細胞からの進行を追跡するために、骨転移に関連した研究目的の広い範囲のために有用である可能性があります微小転移。特に、我々はIIA注射は、癌細胞と骨微小環境における周囲のセルの様々なタイプの間の相互作用を可視化することを可能にすることを実証しました。
Protocol
Representative Results
Discussion
唯一の腸骨動脈は、癌細胞に注入の対象ではありますが、私たちは、周囲の組織からの両方の腸骨静脈と動脈の分離をお勧めします、とのバンドルとして一緒にそれらを持ち上げるために。静脈と動脈が広く互いに接触するためである、と静脈血管壁が薄く、壊れやすいです。がん細胞のみを動脈に注入されているがため、成功した注射のために、それは、一緒に2つの容器を保持するために…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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
| 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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