Abstract
転移は、癌関連の罹患率および死亡率の主要な原因です。転移は多段階プロセスであり、その複雑さのために、転移性播種と成長を支配する正確な細胞および分子プロセスはまだとらえどころのないです。ライブイメージングは、細胞とその微小環境の動的なと空間の相互作用の可視化を可能にします。固形腫瘍は、一般的に肺に転移します。しかし、肺の解剖学的位置は、生体イメージングへの挑戦を提起します。このプロトコルは、肺転移内の腫瘍細胞とその周囲の間質間の動的相互作用の ex vivoライブイメージングのための比較的単純かつ迅速な方法を提供します。この方法を使用して、癌細胞の運動性ならびにそれらの微小環境中の癌細胞と間質細胞の間の相互作用は、いくつかの時間をリアルタイムで可視化することができます。トランスジェニック蛍光レポーターマウスを用いて、蛍光細胞株は、注射可能な蛍光標識分子および/または抗体は、肺の微小環境の複数のコンポーネントは、血管及び免疫細胞のように、可視化することができます。異なる細胞型の画像に、迅速な、四色の画像取得との長期の連続撮影を可能にする回転ディスク共焦点顕微鏡が用いられています。複数の位置と焦点面上で収集した画像からコンパイルタイムラプスムービーは、少なくとも4時間のライブ転移性および免疫細胞間の相互作用を示しています。この技術はさらに、化学療法または標的療法を試験するために使用することができます。また、この方法は、肺の微小環境に影響を及ぼし得る他の肺関連病理の研究のために適合させることができます。
Materials
| Name | Company | Catalog Number | Comments |
| MMTV-PyMT/FVB mice | Jackson Laboratory | 2374 | Female mice |
| ACTB-ECFP/FVB mice | UCSF Werb lab | Female mice | |
| c-fms-EGFP/FVB mice | UCSF Werb lab | Female mice | |
| FVB mice | Jackson Laboratory | 1800 | Female mice |
| GFP+ VO-PyMT cells | UCSF Werb lab | ||
| 70,000 kDa Dextran, rhodamine-conjugated | Invitrogen | D1818 | Dilute to 4mg/ml in 1 x PBS and store at -20 °C. Use 0.4 mg per animal. |
| 10,000 kDa Dextran, Alexa Fluor 647 conjugated | Invitrogen | D22914 | Dilute to 4mg/ml in 1 x PBS and store at -20 °C. Use 0.4 mg per animal. |
| Anti-mouse Gr-1 antibody Alexa Fluor 647 | UCSF Monoclonal antibody core | Stock 1mg/ml. Use 7 ug per animal. | |
| Anesthetic | Anesthesia approved by IACUC, used for anesthesia and/or euthanesia | ||
| 1X PBS | UCSF cell culture facility | ||
| PBS, USP sterile | Amresco INC | K813-500ML | Ultra pure grade for i.v. injection |
| Styrofoam platform | Will be used as dissection board | ||
| Fine scissors sharp | Fine Science Tools | 14060-11 | |
| Forceps | Roboz Surgical Store | RS-5135 | |
| Hot bead sterilizer | Fine Science Tools | 18000-45 | Turn ON 30min before use |
| Air | UCSF | ||
| Oxygen | UCSF | ||
| Carbon dioxide | UCSF | ||
| 1 mL syringe without needle | BD | 309659 | |
| 27 G x 1/2 needle | BD | 305109 | for i.v. injection |
| 20 G x 1 needle, short bevel | BD | 305178 | |
| Low-melting-temperature agarose | Lonza | 50111 | To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 °C water bath until used for inflation. |
| RPMI-1640 medium without phenol red | Life Technologies | 11835-030 | |
| 24 well Imaging plate | E&K scientific | EK-42892 | |
| Glass cover slides, 15 mm | Fisher Scientific | 22-031-144 | |
| Digital CO2 and temperature controller | Okolab | DGTCO2BX | http://www.oko-lab.com |
| Climate chamber | Okolab | http://www.oko-lab.com | |
| Cell Observer spinning disk confocal microscope | Zeiss | ||
| Zen software | Zeiss | ||
| Inverted microscope | Carl Zeiss Inc | Zeiss Axiovert 200M | |
| ICCD camera | Stanford Photonics | XR-Mega-10EX S-30 | |
| Spinning disk confocal scan-head | Yokogawa Corporation | CSU-10b | |
| Imaris | Bitplane | ||
| mManager | Vale lab, UCSF | Open-source software |
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