Udvidet Time-lapse Intravital Imaging af Real-time flercellede Dynamics i Tumor mikromiljø
Summary
Denne protokol beskriver anvendelsen af multifoton mikroskopi til at udføre længere tid-lapse billeddannelse af multicellulære interaktioner i realtid, in vivo ved enkelt celle opløsning.
Abstract
In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.
Introduction
Udbredelse af tumorceller fra primære brysttumor har vist sig at inddrage ikke kun tumorceller, men vært stromale celler, herunder makrofager og endotelceller. Endvidere tumorvaskulatur er unormal med forøget permeabilitet 1. Således bestemme, hvor tumorceller, makrofager og endotelceller interagerer at mediere vaskulær permeabilitet og tumorcelle intravasation i den primære tumor mikromiljø er vigtig for forståelsen metastase. Forståelse kinetikken af vaskulær permeabilitet, tumor celle intravasation og den underliggende signalering mekanisme flercellede interaktioner i tumoren mikromiljø kan give afgørende oplysninger i udvikling og administration af anti-behandlinger mod kræft.
Det primære middel til at studere tumor vaskulær permeabilitet in vivo har været målingen af ekstravaskulære farvestoffer, såsom Evans blue 2, højmolekylære dextraner (155 kDa)3 og fluorophor eller radiotracer-konjugerede proteiner (herunder albumin) 4 ved faste tidspunkter efter injektion af farvestoffet. Forbedringer i mikroskopi, har dyremodeller og fluorescerende farvestoffer aktiveret visualisering af cellulære processer og vaskulær permeabilitet i levende dyr ved intravital mikroskopi 5.
Levende dyr billeddannelse med købet af statiske billeder eller kort tid-lapse-sekvenser over flere tidspunkter ikke giver mulighed for det fuldstændig forståelse af dynamiske begivenheder i tumoren mikromiljø 6,7. Faktisk statisk billede erhvervelse i løbet af 24 timer viste, at tumorvaskulatur er utæt, men dynamikken i vaskulær permeabilitet blev ikke observeret 6. Således extended kontinuert tid-lapse billeddannelse op til 12 timer indfanger kinetikken for dynamiske begivenheder i tumormikromiljøet.
Denne protokol beskriver anvendelsen af extended time-lapse multiphotpå intravital mikroskopi for at studere dynamiske flercellede begivenheder i tumormikromiljøet. Flere celletyper i tumormikromiljøet er mærket med injicerbare farvestoffer eller ved at anvende transgene dyr, der udtrykker fluorescerende proteiner. Ved hjælp af en halevene kateter kan injiceres vaskulære farvestoffer eller proteiner efter begyndelsen af billeddannelse til at erhverve kinetiske data af multicellulære begivenheder i tumormikromiljøet. For levende celler den brysttumor er eksponeret gennem den kirurgiske forberedelse af en hudlap. Billeder er erhvervet i op til 12 timer ved hjælp af en multifoton mikroskop udstyret med flere fotomultiplikatorrør (PMT) detektorer 8. Ved hjælp af passende filtre, en subtraktion algoritme giver 4 PMT-detektorer til at erhverve 5 fluorescerende signaler i tumor mikromiljø samtidigt 9. Høj opløsning multifoton intravital mikroskopi indfanger enkelt celle opløsning billeddannelse af tumor-stroma interaktioner i tumormikromiljøet, hvilket fører til en bedre understanding af vaskulær permeabilitet og tumorcelle intravasation 10-13. Specifikt udvidede intravital imaging afslørede stærkt lokaliseret, forbigående vaskulær permeabilitet hændelser, der forekommer selektivt ved steder for interaktion mellem en tumorcelle, en makrofag og en endotelcelle (defineret som tumormikromiljøet af metastase, TMEM 14) 13. Endvidere tumorcelle intravasation forekommer kun på TMEM og er rumligt og tidsligt korreleret med vaskulær permeabilitet 13. Enkelt celle opløsning af dynamikken i disse begivenheder blev muliggjort gennem anvendelse af længere tid-lapse multifoton mikroskopi af fluorescens-mærkede celler i tumor mikromiljø.
Protocol
Representative Results
Discussion
Cellulære interaktioner, der forekommer spontant i tumormikromiljøet kan føre til ændringer i tumorcellemotilitet og intravasation. Høj opløsning intravital billeddannelse af levende tumorvæv tillader visualisering af multi-cellulære dynamik, der kan være meget forbigående 10,13,24. End-point in vivo assays eller time-lapse billeder erhvervet med diskrete tidspunkter kan give væsentlige oplysninger om molekylære mekanismer i processer i tumor mikromiljø. Intravital billeddannende unders?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev støttet af det amerikanske forsvarsministerium Breast Cancer Research Program under award nummer (ASH, W81XWH-13-1-0010), NIH CA100324, PPG CA100324, og den integrerede Imaging Program.
Materials
| 155 kDa dextran-tetramethylrhodamine isothiocyanate | Sigma Aldrich | T1287 | reconstitute at 20 mg/mL in 1 X PBS |
| 70 kDa dextran-Texas Red | Life Technologies | D-1830 | reconstitute at 10 mg/mL in 1 X PBS |
| 10 kDa dextran-fluorescein isothyocyanate | Sigma Aldrich | FD10S | reconstitute at 20 mg/mL in 1 X PBS |
| Qdot 705 ITK Amino (PEG) Quantum Dots | Life Technologies | Q21561MP | Dilute 25 uL in 175 uL of 1 X PBS for injection |
| MMTV-PyMT mice | Jackson Laboratory | 2374 | |
| Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP) | Jackson Laboratory | 26051 | |
| Csf1r-EGFP mice | Jackson Laboratory | 18549 | |
| 1 x PBS | Life Technologies | ||
| Isoethesia (isoflurane) | Henry Schein Animal Health | 50033 | 250 mL |
| Oxygen | AirTech | ||
| 1 mL syringe, tuberculin slip tip | BD | 309659 | |
| 30G x 1 (0.3 mm X 25 mm) needle | BD | 305128 | |
| Polyethylene micro medical tubing | Scientific Commodities Inc | BB31695-PE/1 | 0.28 mm I.D. X 0.64 mm O.D. |
| Microscope coverglass | Corning | 2980-225 | thickness 1.5, 22 X 50 mm |
| MouseOx oximeter, software and sensors | STARR Life Sciences | ||
| Laboratory tape | Fisher Scientific | 159015R | |
| soft rubber pad | McMaster-Carr | 8514K62 | Ultra-Soft Polyurethane Film, 3/16” Thick, 12" x 12", 40 Oo Durometer, Plain Back |
| hard rubber pad | McMaster-Carr | 8568K615 | High-Strength Neoprene Rubber Sheet 1/4" Thick, 12" X 12", 50A Durometer, |
| Microscope | Olympus | The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20x 1.05NA objective lens. | |
| 7-Punch set | McMaster-Carr | 3429A12 | , 1/4" to 1" Hole Diameter, for Hammer-Driven Hole Punch, |
References
- Gerlowski, L. E., Jain, R. K. Microvascular permeability of normal and neoplastic tissues. Microvasc Res. 31 (3), 288-305 (1986).
- Wang, H. -. L., Lai, T. W. Optimization of Evans blue quantitation in limited rat tissue samples. Sci Rep. 4, (2014).
- Dvorak, H. F. Rous-Whipple Award Lecture. How tumors make bad blood vessels and stroma. Am J Pathol. 162 (6), 1747-1757 (2003).
- Nagy, J. A., et al. Permeability properties of tumor surrogate blood vessels induced by VEGF-A. Lab Invest. 86 (8), 767-780 (2006).
- Egawa, G., et al. Intravital analysis of vascular permeability in mice using two-photon microscopy. Sci Rep. 3, (2013).
- Yuan, F., et al. Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res. 55 (17), 3752-3756 (1995).
- Dellian, M., Yuan, F., Trubetskoy, V. S., Torchilin, V. P., Jain, R. K. Vascular permeability in a human tumour xenograft: molecular charge dependence. Br J Cancer. 82 (9), 1513-1518 (2000).
- Entenberg, D., et al. Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat Protocols. 6 (10), 1500-1520 (2011).
- Entenberg, D., et al. Imaging tumor cell movement in vivo. Curr Protoc Cell Biol. Chapter. 19, (2013).
- Patsialou, A., et al. Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo. cell migration in human breast tumors. Intravital. 2 (2), e25294 (2013).
- Gligorijevic, B., Bergman, A., Condeelis, J. Multiparametric classification links tumor microenvironments with tumor cell phenotype. PLoS Biol. 12 (11), e1001995 (2014).
- Roussos, E. T., et al. Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer. J Cell Sci. 13, 2120-2131 (2011).
- Harney, A. S., et al. Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA). Cancer Discov. 5 (9), 932-943 (2015).
- Robinson, B. D., et al. Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination. Clin Cancer Res. 15, 2433-2441 (2009).
- Sasmono, R. T., et al. A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood. 101 (3), 1155-1163 (2003).
- Ovchinnikov, D. A., et al. Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice. J Leukcocyte Biol. 83 (2), 430-433 (2008).
- Liu, H., et al. Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models. Proc Natl Acad Sci U S A. 107 (42), 18115-18120 (2010).
- Nakasone, E. S., Askautrud, H. A., Egeblad, M. Live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer. J Vis Exp. (73), (2013).
- Weis, S., Cui, J., Barnes, L., Cheresh, D. Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis. J Cell Biol. 167 (2), 223-229 (2004).
- Wyckoff, J., Gligorijevic, B., Entenberg, D., Segall, J., Condeelis, J. High-Resolution Multiphoton Imaging of Tumors. In Vivo. Cold Spring Harb Protoc. (10), (2011).
- Lathia, J. D., et al. Direct in vivo. evidence for tumor propagation by glioblastoma cancer stem cells. PLoS One. 6 (9), e24807 (2011).
- Nakasone, E. S., et al. Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance. Cancer Cell. 21 (4), 488-503 (2012).
- Dreher, M. R., et al. Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst. 98 (5), 335-344 (2006).
- Wyckoff, J. B., et al. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res. 67 (6), 2649-2656 (2007).
- Chittajallu, D. R., et al. In vivo. cell-cycle profiling in xenograft tumors by quantitative intravital microscopy. Nat Methods. 12 (6), 577-585 (2015).
- Kedrin, D., et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods. 5 (12), 1019-1021 (2008).
- Yano, S., et al. Spatial-temporal FUCCI imaging of each cell in a tumor demonstrates locational dependence of cell cycle dynamics and chemoresponsiveness. Cell Cycle. 13 (13), 2110-2119 (2014).
- Yang, M., Jiang, P., Hoffman, R. M. Whole-body subcellular multicolor imaging of tumor-host interaction and drug response in real time. Cancer Res. 67 (11), 5195-5200 (2007).
- Manning, C. S., et al. Intravital imaging reveals conversion between distinct tumor vascular morphologies and localized vascular response to Sunitinib. Intravital. 2 (1), 24790 (2013).
- Alexander, S., Koehl, G. E., Hirschberg, M., Geissler, E. K., Friedl, P. Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model. Histochem Cell Biol. 130 (6), 1147-1154 (2008).
- Brown, E. B., et al. In vivo. measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat Med. 7 (7), 864-868 (2001).
