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

Undersøgelse af tumorcellespredning fra lungemetastaser

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.

Forfatter Spotlight: Afkodning af metastase-til-metastase-såning ved hjælp af en ny in vivo-teknik til sporing af spredning af brystkræft 

Sporing af tumorcelleformidling fra lungemetastaser ved hjælp af fotokonvertering

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  

Kirurgisk protokol til selektiv fotokonvertering af lungemetastaser

Efter at have forberedt musen til operation, skal du identificere målområdet, syv millimeter til venstre for brystbenet og ca. syv millimeter over subkostalmargenen. Løft huden over det identificerede område ved hjælp af tang og lav et 10 millimeter cirkulært snit ved hjælp af skarp mikrodissekerende saks for kun at skære huden. Fjern derefter blødt væv over brystkassen og cauterize eventuelle større blodkar i begge ender ved hjælp af en cautery pen.Løft det sjette eller syvende ribben med tang, og brug et enkelt blad af den stumpe mikrodissekerende saks til at gennembore den interkostale muskel. Drej nu forsigtigt saksen og lav et snit på ca. 10 millimeter i det interkostale rum, samtidig med at man undgår kontakt med lungevævet og ribbenene. Frigør trykluft ved håndleddet eller hånden forsigtigt for at bestemme den passende strømningsstyrke og rydde dysen for snavs.Påfør derefter korte udbrud af luft ved snittet for at undgå at forårsage lungeskade. Med tangen løftes den sjette eller syvende ribben og indsættes tilbagetrækningen i det interkostale snit med knive parallelt med brystvæggen. Slip derefter håndtaget forsigtigt for at lade retraktoren åbne og afsløre lungen gradvist.Anvend nu to til tre dråber steril PBS opvarmet til 37 grader på det udsatte lungevæv for at forhindre det i at tørre. Overlejr et stykke aluminiumsfolie med en lille udskæring over musen, og placer den direkte over det udsatte lungevæv. Sørg for, at udskæringen kun viser den åbne thorax og det nærliggende væv til målrettet lungefotokonvertering.Placer derefter en kasse med en udskæring over musen og aluminiumsfolien, og sørg for, at udskæringen flugter med den udsatte lunge. Og indstil fotokonverteringslampen direkte over udskæringen på kassen. Aktivér derefter fotokonverteringslampen for at belyse det udsatte lungevæv i et kontinuerligt tidsrum på seks minutter.Efter fotokonverteringen skal du deaktivere lampen og rydde lampeboksen og aluminiumsfoliemasken væk fra musen. Adskil lungen fra brystvæggen ved at udlede luft ved håndleddet, og udlad derefter luft ved brystsnittet. Tag godt fat i tilbagetrækningshåndtaget, og klem det for at lukke knivene.Manøvrer forsigtigt den lukkede retraktor ud af det interkostale rum, og sørg for ikke at komme i kontakt med lungevævet. Brug derefter 5-0 silkesuturen til at lukke det interkostale snit med et simpelt kontinuerligt suturmønster, hvilket sikrer, at ribben på begge sider af snittet er inkorporeret. Træk suturen fast for at sikre, at sårkanterne justeres, hvilket genopretter brystvæggens integritet.Fastgør suturen ved at knytte enden fire gange. Trim suturhalerne så tæt på knuden som muligt, og luk huden ved hjælp af kirurgiske hæfteklammer. For at fjerne overskydende luft fra brysthulen skal du indsætte en milliliter sprøjte med en 28 gauge nål under xiphoid-processen.Træk sprøjten tilbage til ca. en milliliter. Fjern sprøjten fra musen og udlad luften. Billeder af eksponeret Dendra2, der udtrykker lungemetastaser før eksponering for blåt lys, vises.Eksponering af Dendra2, der udtrykker lungemetastaser for blåt lys, viste, at seks minutters eksponering producerede det lyseste signal i den fotokonverterede kanal i både ex vivo- og in vivo-prøver. Fotokonverterede og ikke-fotokonverterede celler blev identificeret i alle tre vævstyper fra mus, der blev opereret med eksponering for blåt lys. Kun ikke-fotokonverterede tumorceller blev fundet i væv fra mus, der gennemgik operation uden eksponering for blåt lys.

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 ...