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

Esame della disseminazione delle cellule tumorali da metastasi polmonari

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.

Autore in primo piano: Decodifica della semina da metastasi a metastasi utilizzando una nuova tecnica in vivo per monitorare la diffusione del cancro al seno 

Monitoraggio della disseminazione delle cellule tumorali dalle metastasi polmonari utilizzando la fotoconversione

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  

Protocollo chirurgico per la fotoconversione selettiva delle metastasi polmonari

Dopo aver preparato il topo per l'intervento chirurgico, identificare l'area bersaglio, sette millimetri a sinistra dello sterno e circa sette millimetri sopra il margine sottocostale. Sollevare la pelle sopra l'area identificata usando una pinza e praticare un'incisione circolare di 10 millimetri utilizzando forbici affilate per micro dissezione per tagliare solo la pelle. Quindi rimuovere i tessuti molli sopra la gabbia toracica e cauterizzare i principali vasi sanguigni su entrambe le estremità utilizzando una penna per cauterizzazione.Sollevare la sesta o settima costola con una pinza e utilizzare una singola lama delle forbici da microdissezione smussate per perforare il muscolo intercostale. Ora ruotare delicatamente le forbici e praticare un'incisione di circa 10 millimetri nello spazio intercostale evitando il contatto con il tessuto polmonare e le costole. Rilasciare delicatamente l'aria compressa al polso o alla mano per determinare l'intensità del flusso adatta e liberare l'ugello dai detriti.Quindi applicare brevi getti d'aria sull'incisione per evitare di causare lesioni polmonari. Con la pinza, sollevare la sesta o settima costola e inserire il divaricatore nell'incisione intercostale con lame parallele alla parete toracica. Quindi rilasciare con cautela la maniglia per consentire l'apertura del divaricatore e rivelare gradualmente il polmone.Ora applicare due o tre gocce di PBS sterile riscaldato a 37 gradi sul tessuto polmonare esposto per evitare che si secchi. Sovrapponi un pezzo di foglio di alluminio con un piccolo ritaglio sopra il topo, posizionandolo direttamente sul tessuto polmonare esposto. Assicurati che il ritaglio mostri solo il torace aperto e il tessuto vicino per una fotoconversione polmonare mirata.Quindi posiziona una scatola con un ritaglio sopra il mouse e un foglio di alluminio, assicurandoti che il ritaglio sia allineato con il polmone esposto. E posiziona la lampada di conversione delle foto direttamente sopra il ritaglio sulla scatola. Quindi attivare la lampada di conversione fotografica per illuminare il tessuto polmonare esposto per un intervallo continuo di sei minuti.Dopo la conversione delle foto, disattiva la lampada e allontana la scatola della lampada e la maschera in foglio di alluminio dal mouse. Separare il polmone dalla parete toracica scaricando l'aria dal polso, quindi scaricare l'aria dall'incisione toracica. Afferrare saldamente la maniglia del divaricatore e premerla per chiudere le lame.Manovrare con cautela il divaricatore chiuso fuori dallo spazio intercostale, assicurandosi di non entrare in contatto con il tessuto polmonare. Quindi utilizzare la sutura di seta 5-0 per chiudere l'incisione intercostale con un semplice modello di sutura continuo, assicurandosi che la costola su entrambi i lati dell'incisione sia incorporata. Tirare con decisione la sutura per assicurarsi che i bordi della ferita siano allineati, ristabilendo l'integrità della parete toracica.Fissare la sutura annodando l'estremità quattro volte. Taglia le code di sutura il più vicino possibile al nodo e chiudi la pelle usando graffette chirurgiche. Per rimuovere l'aria in eccesso dalla cavità toracica, inserire una siringa da un millilitro con un ago calibro 28 sotto il processo xifoideo.Riportare la siringa a circa un millilitro. Rimuovere la siringa dal mouse e scaricare l'aria. Vengono mostrate immagini di metastasi polmonari esprimenti Dendra2 esposte prima dell'esposizione alla luce blu.L'esposizione alla luce blu delle metastasi polmonari che esprimono Dendra2 ha mostrato che l'esposizione di sei minuti ha prodotto il segnale più luminoso nel canale fotoconvertito sia nei campioni ex vivo che in vivo. Le cellule fotoconvertite e non fotoconvertite sono state identificate in tutti e tre i tipi di tessuto di topi sottoposti a intervento chirurgico con esposizione alla luce blu. Solo cellule tumorali non fotoconvertite sono state trovate nei tessuti dei topi che hanno subito un intervento chirurgico senza esposizione alla luce blu.

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 ...