Ex vivo Live-Imaging van Lung metastase en hun Microenvironment
Summary
Beschrijven we een betrekkelijk eenvoudige werkwijze voor ex vivo beeldvorming van levende het tumorcel-stroma interacties binnen longmetastasen, gebruik fluorescente reporters in muizen. Gebruik draaiende schijf confocale microscopie Deze techniek maakt visualisatie van levende cellen gedurende ten minste 4 uur en kan worden aangepast om andere inflammatoire longaandoeningen bestuderen.
Abstract
Metastase is een belangrijke oorzaak van kanker-gerelateerde morbiditeit en mortaliteit. Metastase is een multi-step proces en als gevolg van de complexiteit, de precieze cellulaire en moleculaire processen die metastatische verspreiding en groei regeren zijn nog steeds ongrijpbaar. Levende beeldvorming maakt visualisatie van de dynamische en ruimtelijke interactie van cellen en hun micromilieu. Vaste tumoren vaak uitzaaien naar de longen. De anatomische locatie van de longen vormt een uitdaging voor intravitale beeldvorming. Dit protocol verschaft een relatief eenvoudige en snelle werkwijze voor ex vivo beeldvorming van levende de dynamische interacties tussen tumorcellen en de omliggende stroma binnen longmetastasen. Door deze methode kan de beweeglijkheid van kankercellen en interacties tussen kankercellen en stromale cellen in hun micromilieu worden gevisualiseerd in realtime enkele uren. Door het gebruik van transgene fluorescerende reporter muizen, een tl-cellijn, injecteerbare fluorescent gelabeldemoleculen en / of antilichamen, verschillende onderdelen van de long micro kan worden gevisualiseerd, zoals bloedvaten en immuuncellen. Om het beeld verschillende celtypen, is een draaiende schijf confocale microscoop die langdurige continue beeldvorming met een snelle, vier kleuren beeldacquisitie maakt gebruikt. Time-lapse filmpjes samengesteld uit beelden verzameld over meerdere posities en focale vliegtuigen tonen interactie tussen live-metastatische en immuuncellen gedurende ten minste 4 uur. Deze techniek kan verder worden gebruikt voor chemotherapie of gerichte therapie testen. Bovendien kan deze werkwijze worden aangepast voor de studie van andere long-gerelateerde pathologieën die de long micro beïnvloeden.
Introduction
The deadliest aspect of cancer is metastasis, which accounts for more than 90% of cancer-related morbidity and mortality1. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular mechanisms that govern metastatic dissemination and growth are still elusive. To metastasize, tumor cells in the primary tumor must detach from their neighboring cells and basement membrane, cross through the extracellular matrix, intravasate, travel via blood or lymphatic vessels, extravasate at the secondary site, and finally, survive and establish secondary tumors. In addition to the properties of the tumor cells, the contribution from the microenvironment, which includes the adjacent stroma along with the normal counterparts of the cancer cells, is crucial for the seeding and establishment of metastatic lesions2.
Traditional methods to study metastatic seeding and growth examine static states, as tissues are excised and sectioned for histology. These data only generate a snapshot of this highly dynamic process. Although some useful information can be gained from these studies, the complicated process by which tumor and stromal cells interact during metastatic formation cannot be adequately assessed by these methods. Furthermore, it is not possible to gain insights into tumor or stromal cell migration patterns, which are important in establishing a colony at the distant site. In order to effectively study the metastatic process, it is essential to visualize various interactions between cancer cells and their microenvironment in a continuous manner and at real time.
The lung is a common site for metastases from solid tumors as breast, colorectal, pancreatic cancer, melanoma and sarcoma3. Intravital imaging was previously used to study cell-cell interaction in various primary tumor and metastatic models4,5. Methods of lung imaging in mice, including intravital imaging, lung section imaging, and an ex vivo pulmonary metastasis assay have been published6–9. Intravital imaging of mouse lungs utilizes a thoracic suction window to stabilize the lungs6. This method is used for time-lapse imaging of the lung microcirculation and alveolar spaces. The anatomical location of the lungs poses a challenge to intravital imaging. In order to access the lungs, the chest cavity must be opened which leads to loss of negative pressure and collapsed lungs. This method only allows the visualization of a small part of the lungs and is technically demanding; an unnecessary complication in studies that examine processes that are independent of blood flow. Moreover, this method also requires gating out movement caused by breathing. This is done either by collecting images between breaths or during post image acquisition analyses10. The alternative ex vivo lung section imaging provides stability and depth, and also prepares lung parenchyma for immunostaining7. However, the lengthy sectioning process leads to an extensive delay between the time of animal sacrifice and the start of the imaging session. Moreover, the process of sectioning a mouse lung causes considerable amount of cell death8, thus interfering with the quality and quantity of imaging samples and perhaps needlessly altering tumor-stroma interactions. In order to technically bridge between the methods of intravital imaging and lung section imaging, while exploiting the advantages of the two techniques, a relatively fast and easy method for ex vivo lung imaging was developed. This method was achieved by imaging of non-sectioned whole lung lobes. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours.
Protocol
Representative Results
Discussion
Dit manuscript beschrijft een gedetailleerde methode voor ex vivo live-beeldvorming van de long metastase in muismodellen van metastase. Deze beeldprotocol een directe visualisatie van de dynamische en ruimtelijke tumorcel-stroma interacties binnen de long micro. Het is een relatief eenvoudige en snelle methode die betrouwbare beeldvorming van longmetastase gedurende ten minste 4 uur mogelijk maakt. Films verkregen uit deze experimenten kan worden gebruikt om dynamische processen celmotiliteit en cellulaire int…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank Nguyen H. Nguyen for her technical help and Audrey O’Neill for support with the Zeiss Cell Observer spinning-disk confocal microscope. This work was supported by a Department of Defense postdoctoral fellowship (W81XWH-11-01-0139) and the Weizmann Institute of Science-National Postdoctoral Award Program for Advancing Women in Science (to V.P.).
Materials
| 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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