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

Examen de la dissémination des cellules tumorales à partir de métastases pulmonaires

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.

Pleins feux sur l’auteur : Décoder l’ensemencement de métastases à métastases à l’aide d’une nouvelle technique in vivo pour suivre la propagation du cancer du sein 

Suivi de la dissémination des cellules tumorales à partir de métastases pulmonaires à l’aide de la photoconversion

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  

Protocole chirurgical pour la photoconversion sélective des métastases pulmonaires

Après avoir préparé la souris à la chirurgie, identifiez la zone cible, à sept millimètres à gauche du sternum et à environ sept millimètres au-dessus du bord sous-costal. Soulevez la peau sur la zone identifiée à l’aide d’une pince et faites une incision circulaire de 10 millimètres à l’aide de ciseaux de micro-dissection tranchants pour couper uniquement la peau. Retirez ensuite les tissus mous au-dessus de la cage thoracique et cautérisez les principaux vaisseaux sanguins aux deux extrémités à l’aide d’un stylo de cautérisation.Élevez la sixième ou la septième côte avec une pince et utilisez une seule lame des ciseaux de micro-dissection émoussés pour percer le muscle intercostal. Maintenant, faites tourner doucement les ciseaux et faites une incision d’environ 10 millimètres dans l’espace intercostal tout en évitant le contact avec le tissu pulmonaire et les côtes. Libérez délicatement l’air comprimé au niveau du poignet ou de la main pour déterminer la force d’écoulement appropriée et débarrasser la buse des débris.Appliquez ensuite de courtes bouffées d’air sur l’incision pour éviter de causer des lésions pulmonaires. À l’aide de la pince, soulevez la sixième ou la septième côte et insérez l’écarteur dans l’incision intercostale avec des lames parallèles à la paroi thoracique. Relâchez ensuite la poignée avec précaution pour laisser l’écarteur s’ouvrir et révéler le poumon progressivement.Appliquez maintenant deux à trois gouttes de PBS stérile chauffé à 37 degrés sur le tissu pulmonaire exposé pour l’empêcher de se dessécher. Superposez un morceau de papier d’aluminium avec une petite découpe sur la souris, en le positionnant directement sur le tissu pulmonaire exposé. Assurez-vous que la découpe ne montre que le thorax ouvert et le tissu voisin pour une conversion ciblée de la photo pulmonaire.Ensuite, placez une boîte avec une découpe sur la souris et du papier d’aluminium, en vous assurant que la découpe s’aligne avec le poumon exposé. Et placez la lampe de conversion photo directement au-dessus de la découpe sur la boîte. Activez ensuite la lampe de photoconversion pour éclairer le tissu pulmonaire exposé pendant une période continue de six minutes.Après la conversion de la photo, éteignez la lampe et retirez le boîtier de la lampe et le masque en papier d’aluminium de la souris. Séparez le poumon de la paroi thoracique en évacuant l’air au niveau du poignet, puis évacuez l’air au niveau de l’incision thoracique. Saisissez fermement la poignée de l’enrouleur et pressez-la pour fermer les lames.Manœuvrez avec précaution l’écarteur fermé hors de l’espace intercostal, en veillant à ne pas entrer en contact avec le tissu pulmonaire. Utilisez ensuite la suture en soie 5-0 pour fermer l’incision intercostale avec un simple motif de suture continu, en veillant à ce que la côte des deux côtés de l’incision soit incorporée. Tirez fermement sur la suture pour vous assurer que les bords de la plaie s’alignent, rétablissant ainsi l’intégrité de la paroi thoracique.Fixez la suture en nouant l’extrémité quatre fois. Coupez les queues de suture aussi près que possible du nœud et fermez la peau à l’aide d’agrafes chirurgicales. Pour éliminer l’excès d’air de la cavité thoracique, insérez une seringue d’un millilitre avec une aiguille de calibre 28 sous le processus xiphoïde.Ramenez la seringue à environ un millilitre. Retirez la seringue de la souris et évacuez l’air. Des images de Dendra2 exposé exprimant des métastases pulmonaires avant l’exposition à la lumière bleue sont montrées.L’exposition de Dendra2 exprimant des métastases pulmonaires à la lumière bleue a montré qu’une exposition de six minutes produisait le signal le plus brillant dans le canal photoconverti dans les échantillons ex vivo et in vivo. Des cellules photoconverties et non photoconverties ont été identifiées dans les trois types de tissus de souris ayant subi une intervention chirurgicale avec exposition à la lumière bleue. Seules des cellules tumorales non photoconverties ont été trouvées dans les tissus de souris qui ont subi une intervention chirurgicale sans exposition à la lumière bleue.

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 ...