Name: Surgical Protocol for the Selective Photoconversion of Lung Metastases
Uploaded: 2023-07-07T14:40:00
Description: Watch this Scientific Journal Video about Surgical Protocol for the Selective Photoconversion of Lung Metastases at JoVE.com

surgical protocol for the selective photoconversion of lung metastases

肺転移からの腫瘍細胞播種の調査

Metastasis is the leading cause of cancer-related deaths1.&#160;Metastatic cancer arises when cells from the primary tumor disseminate throughout the body and proliferate into clinically detectable tumors in distant organs2,3.
Although metastasis is commonly thought of as a unidirectional process wherein tumor cells disseminate from the primary tumor and colonize distant organs4, increasing clinical and experimental evidence suggests that a more complex, multi-directional process is at play. It has been shown that circulating tumor cells can reseed the primary tumor (if still in place)5,6,7,8,9, and tumor cells from existing metastatic foci can travel to tertiary sites and give rise to new lesions10,11,12,13. Indeed, evidence from recent genomic analyses suggests that some metastatic lesions arise not from the primary tumor, but from other metastases-a phenomenon known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding&#34;14,15,16. Metastasis-to-metastasis seeding can perpetuate the disease process even after removal of the primary tumor, increasing metastatic burden, and decreasing patient quality of life and survival. Therefore, understanding the processes behind metastasis-to-metastasis seeding is crucial to refining treatment strategies for patients with metastatic disease.
Despite the potentially severe clinical implications, little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Human studies are limited by a paucity of clinical samples. Clinical resection and biopsy of metastatic lesions are uncommon, as is the biopsy of seemingly healthy organs, where single disseminated tumor cells may lurk. This means that human studies are typically only possible using autopsy samples from individuals whose primary tumors are either still in place or were previously resected but are still available to researchers. When such samples are available, lineage analyses of cancer progression must be performed using sequencing methods14. However, bulk sequencing of matched primary tumors and metastases does not have the sensitivity needed for comprehensive lineage tracing. For instance, bulk sequencing of one lesion may reveal a subclone that is undetectable in any of its matched lesions. In this case, one would be unable to determine the origin of this subclone. It may have been present in the primary tumor or another metastasis at a frequency below the limit of detection, or it may have arisen after the initial colonization of the metastatic lesion it was found in. Single-cell sequencing provides increased sensitivity, but its high cost limits the large-scale application of this technique. The retrospective nature of these studies also means that they provide limited insight into transient metastatic events and the disease landscape at different time points.
In animal models, recent technological advances now allow for prospective phylogenetic mapping with high spatial and temporal resolution17,18,19,20. These techniques utilize CRISPR/Cas9 genome editing to engineer cells with an evolving barcode - heritable mutations that accumulate over time. Upon sequencing, the lineage of each cell can be traced based on the mutational profile of its barcode17,18,19,20. Indeed, such technology is already being used to map metastasis-to-metastasis seeding. In a recent paper, Zhang et al. demonstrated that breast and prostate cancer cells in bone metastases redisseminate from the bone to seed secondary metastases in multiple organs21.
While these novel methods have great potential to generate detailed, high-resolution phylogenetic maps of cancer progression, they are highly impractical for those studying the exact timing of metastasis-to-metastasis seeding events and what promotes or prevents them. Filling these knowledge gaps is crucial to refining our understanding and treatment of metastatic cancer, but there is a noticeable lack of technologies to facilitate such studies. To address this need, we recently developed - and present herein - a novel technique that allows us to specifically mark tumor cells via photoconversion in a metastatic site (the lung) and subsequently reidentify them in tertiary organs. Using this technique, we recently showed that breast cancer cells do redisseminate from lung metastases and seed tertiary organs13. This technique may also be used to determine the timing of redissemination events within a narrow window and quantify redisseminated tumor cells, facilitating the study of organotropism of redisseminated cells and what promotes/prevents redissemination.
While photoconversion and locally inducible cre/lox systems that permanently replace one fluorescent protein with another have been previously used to mark and track tumor cells11,22,23, to our knowledge, no approach for spatiotemporal marking of tumor cells has been optimized to target the lung - one of the most common sites of metastasis among men and women diagnosed with any of the 14 most common cancers24. Any cancer cell type and any protocol for lung metastasis generation may be used with our procedure, making it broadly useful for metastasis researchers. All cancer cells used to generate lung metastases should express a photoconvertible or photo-switchable protein, and researchers may choose which protein to use based on their specific needs and resources. In this study, we used 6DT1 breast cancer cells that stably expressed the photoconvertible green-to-red fluorescent protein Dendra2 (6DT1-Dendra2 cells)25 tagged to the histone H2B. We injected 5.0 &#215; 104 6DT1-Dendra2 cells into the fourth mammary fat pad of female Rag2-/- mice. Primary tumors were palpable between 12 and 16 days after injection and were not resected for the duration of the experiment. Spontaneous lung metastases developed between 19 and 26 days after tumor cell injection. Photoconversion surgeries were performed between 26 and 29 days after tumor cell injection. Mice were sacrificed by 72 h post surgery due to lung metastasis burden.

著者スポットライト:乳がんの広がりを追跡するための新しいIn vivo技術を使用した転移間播種の解読 

光変換を用いた肺転移からの腫瘍細胞播種の追跡

Recent evidence suggests that existing metastatic lesions can act as a source of disseminating tumor cells that give rise to new lesions. This process is called metastasis to metastasis seeding and can increase tumor burden and decrease the quality of life and survival of cancer patients. Our method was developed to study metastasis to metastasis seeding and the factors that influence it.This technique has helped our lab establish that breast cancer cells can spread from lung metastases and enabled us to investigate factors that promote or prevent metastasis. These findings may guide future research on slowing the progression of metastatic cancer. The lungs are a common site of metastasis, but very few techniques allow for the study of tumor cell dynamics in vivo.This technique enables the tracking of tumor cells in live mice facilitating the study of lung metastasis specific dissemination pathways and therapies. Metastatic cancer is often an incurable and fatal disease. This technique can be used to study ways to reduce its spread leading to therapies that extend the lives of patients.

Watch this Scientific Journal Video about Surgical Protocol for the Selective Photoconversion of Lung Metastases at JoVE.com

Surgical Protocol for the Selective Photoconversion of Lung Metastases  

肺転移の選択的光変換のための外科的プロトコル

マウスを手術の準備をした後、胸骨の左側7ミリメートル、肋骨下縁の約7ミリメートル上の標的領域を特定します。鉗子を使用して特定された領域に皮膚を持ち上げ、鋭利なマイクロ解剖ハサミを使用して10ミリメートルの円形切開を行い、皮膚のみを切断します。次に、胸郭の軟部組織を取り除き、焼灼ペンを使用して両端の主要な血管を焼灼します。鉗子で6番目または7番目の肋骨を持ち上げ、鈍いマイクロ解剖ハサミの1枚刃を使用して肋間筋を突き刺します。次に、ハサミを静かに回転させ、肺組織と肋骨との接触を避けながら、肋間腔を約10ミリメートル切開します。手首または手で圧縮空気を繊細に放出して、適切な流動強度を決定し、ノズルから破片を取り除きます。次に、肺の損傷を引き起こさないように、切開部に短時間の空気を吹き込みます。鉗子で、6番目または7番目の肋骨を持ち上げ、胸壁に平行なブレードでリトラクターを肋間切開に挿入します。次に、ハンドルを慎重に離して、リトラクターを開き、肺を徐々に露出させます。次に、37度に温めた滅菌PBSを2〜3滴、露出した肺組織に塗布して、乾燥を防ぎます。小さな切り欠きのあるアルミホイルをマウスの上に重ね、露出した肺組織の上に直接配置します。切り欠きには、標的を絞った肺の写真変換のために、開いた胸部と近くの組織のみが表示されていることを確認します。次に、マウスとアルミホイルの上に切り欠きのある箱を配置し、切り欠きが露出した肺に揃うようにします。そして、箱の切り欠きの真上に写真変換ランプをセットします。次に、光変換ランプを作動させて、露出した肺組織を6分間連続して照らします。写真変換後、ランプを無効にし、ランプボックスとアルミホイルマスクをマウスから取り除きます。手首で空気を排出して肺を胸壁から分離し、胸部切開部で空気を排出します。リトラクターハンドルをしっかりとつかみ、握ってブレードを閉じます。閉じたリトラクターを肋間腔から慎重に操作し、肺組織に接触しないようにします。次に、5-0シルク縫合糸を使用して、単純な連続縫合パターンで肋間切開部を閉じ、切開部の両側の肋骨が組み込まれるようにします。縫合糸をしっかりと引っ張って、創傷の端が揃うようにし、胸壁の完全性を再確立します。端を4回結んで縫合糸を固定します。縫合糸の尾部をできるだけ結び目に近づけてトリミングし、外科用ステープルを使用して皮膚を閉じます。胸腔から余分な空気を取り除くには、剣状突起の下に28ゲージの針が付いた1ミリリットルの注射器を挿入します。シリンジを約1ミリリットルに戻します。.マウスからシリンジを取り外し、空気を排出します。青色光曝露前に肺転移を発現するDendra2の画像を示す。肺転移を発現するDendra2を青色光に曝露したところ、6分間の曝露により、ex vivoサンプルとin vivoサンプルの両方で、光変換されたチャネルで最も明るいシグナルが生成されることが示されました。青色光曝露で手術を受けたマウスの3つの組織タイプすべてにおいて、光変換細胞および非光変換細胞が同定された。青色光に曝露せずに手術を受けたマウスの組織には、非光変換された腫瘍細胞のみが見つかりました。

Research

JoVE Journal

Cancer Research

Surgical Protocol for the Selective Photoconversion of Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...