腫瘍の増殖と転移研究のためのトレーサブル記者との乳癌患者由来の異種移植片の標識
Summary
We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.
Abstract
The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients’ tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.
Introduction
外科的に切除腫瘍サンプルは、直接免疫不全マウスに移植された患者由来の腫瘍異種移植片（PDXs）の開発は、標準的な細胞株の異種移植モデルに比べていくつかの利点を提供し、癌研究1,2における大きな進歩を表しています。 PDXsは最初の継代で増殖させ、腫瘍の遺伝的および生物学的特性の最小の変化で連続継代により維持及び拡張することができます。そしてより正確にヒト癌細胞株3-8由来の異種移植片よりも腫瘍の不均一性を反映しています。これらのモデルは、現在広く医薬品開発6,11における前臨床プラットフォームとしておよび癌生物学4,12を研究するための実験的なツールとして、癌治療9,10をパーソナライズするためのプラットフォームとして使用されています。
ほとんどのPDXsは、移植され、実行可能にキャリパーを使用して、時間をかけて腫瘍増殖の測定を可能にする、皮下に伝播されます。しかしながら、転移性疾患はPDXsを使用してモデル化することはより困難でした。具体的には乳癌のために、別の器官への転移能力を有する異種移植片3,5,13を説明したが、転移部位への自発的な普及の頻度が極めて低いです。報告された場合には、転移性の負担の同定および定量は、死後標的臓器の面倒な組織学的検査に依存しています。生物発光を発現する癌細胞株（ルシフェラーゼ、リュック）または蛍光（緑色蛍光タンパク質、GFP）の遺伝子レポーターは、一般的に、脳、肺、心臓内の後の骨、肝臓、尾静脈、大腿骨内および脾臓注射乳癌転移の実験モデルに使用されています14-16。これらのモデルは、原発腫瘍から播種をバイパスするが、それらは臓器向性および転移性のコロニー形成のメカニズムを研究する価値があります。しかし、一次患者の腫瘍とPDXs由来の細胞は、低トランスフェクションまたは形質導入率usinを持つことができますグラム標準的な手順。一つの代替案は、その後、従来の組織培養プロトコルを用いて標識することができ、インビトロ 17 に PDX由来細胞株を確立することです。このアプローチは、しかしながら、細胞株の誘導が困難であり、細胞の表現型を変更することができるため、最もPDXsを標識するのに適していません。ここでは、in vivoイメージングに適したレンチウイルスベクターを用いたPDX-解離腫瘍細胞の形質導入のためのプロトコルを提示します。加えて、我々は、免疫不全マウスにおいて、解離したLUC-GFP標識されたPDX細胞の心臓内注射を使用して実験的転移を説明します。
遺伝子レポーターを発現するレンチウイルスとPDX-解離オルガノイドの形質導入のための基本的なプロトコルは、以前に18を説明してきました。現在のプロトコルでは、ヒト腫瘍細胞を濃縮し、100％の導入効率の近くに取得するために追加の方法を説明するだけでなく、実験的な乳癌を検出するための標識PDXsの使用転移。このプロトコルは、様々な発光と蛍光マーカーと同様に、遺伝子発現（目的の遺伝子の、すなわち 、のshRNAノックダウン）の調節にPDXsの複数の癌種を標識するために適合させることができます。
Protocol
Representative Results
Discussion
Critical steps within the protocol:
The use of high titer lentiviral particles (>108 TU/ml) is a critical step in the success of this protocol, as allows careful control of the media composition during in vitro transduction. While multiple methods for production of high-titer viral particles have been well described18,19; this protocol uses lentiviral particles produced as described in detail at www.kottonla…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Dr. Darrel Kotton at Boston University for providing the pHAGE-EF1aL-dsRed-UBC-GFP-W vector and protocols for high titer lentiviral production used in these studies. This work was funded by DOD BCRP W81XWH-11-1-0101 (DMC), ACS IRG # 57-001-53 (DMC), NCI K22CA181250 (DMC) and R01 CA140985 (CAS).NCI P30CA046934 Center grant supported in vivo imaging and tissue culture cores at University of Colorado AMC.
Materials
| DMEM/F12 (1:1) | Hyclone | SH30023.01 | |
| bFGF | BD Biosciences | 354060 | |
| EGF | BD Biosciences | 354001 | |
| Heparin | Sigma | H4784 | |
| B27 | Gibco/Thermo Fisher | 17504-44 | |
| Anti-fungi-antibiotics | Hyclone | SV30010 | |
| Accumax | Innovative Cell Technologies | AM-105-500 | Digestion Buffer |
| FBS | Atlanta Biologicals | S11550 | |
| HBSS Red Ca++/Mg++ free | Hyclone | SH30031.02 | |
| Hepes | |||
| 10X PBS | Hyclone | SH30258.01 | |
| Cultrex | Cultrex | 3433-005-01 | Basement Matrix Extract (BME) |
| 30C shaker | NewBrunswick Scientific CO. INC | Series 25 Incubator Shaker | |
| 70um filters | Falcon | 7352350 | |
| scalpels | Fisher | 22079690 | |
| Clorhexidine disinfectant | Durvet | NDC# 30798-624-35 | |
| Red blood cell lysis reagent | Sigma | R7757 | |
| Neuraminidase | Sigma | N7885-1UN | |
| EpCAM (CD326+) microbeads* | Miltenyil Biotec | 130-061-101 | |
| Lineage cell depletion Kit, mouse* | Miltenyil Biotec | 130-090-858 | |
| MiniMACS Separator | Miltenyil Biotec | 130-042-102 | |
| Mini MACS Magnetic Stand | Miltenyil Biotec | 130-042-303 | |
| MS Columns | Miltenyil Biotec | 130-042-201 | MS or LS columns can be used, adjust to number of cells. |
| Illumatool Tunable light system | Lightools research | Various | For in vivo fluorescence imaging |
| Xenogen IVIS200 imaging device | Xenogen | Various | For in vivo luminiscence imaging |
| Human Cytokeratin Clone MNF116 Monoclonal antibody | DAKO | M0821 | Pan-cytokeratin |
| Epidermal Growth factor receptor antibody | Cell signaling | 4267S | EGFR |
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