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Developments in preclinical cancer imaging: innovating the discovery of therapeutics

Subjects

Key Points

  • Advanced fluorescence-based imaging, three-dimensional intermediate systems and intravital mouse models can be integrated into the standard drug project operating model (DPOM) to better inform the development and selection of new candidate drugs.

  • Intermediate systems provide initial three-dimensional imaging early in the drug discovery process to support translational cancer research in more physiologically relevant in vitro settings and identify deficient or ineffective drug strategies earlier in the drug discovery pipeline.

  • In vivo advanced imaging techniques can be used to assess more complex questions, such as transient protein–protein interactions or molecular, cell or tissue-specific dynamics in response to drug treatment in live tissue.

  • Biosensors are now providing dynamic and reversible fluorescence-based readouts of drug targeting, allowing drug turnover, clearance and dissociation to be monitored in real-time.

  • Stromal targeting of the tumour microenvironment is a vital aspect of cancer drug development, which can be quantified using advanced imaging techniques, such as second harmonic generation (SHG), third harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM).

  • Longitudinal imaging through intravital imaging windows can give quantitative functional information from repeated, non-invasive imaging and drug endpoint analysis in real-time.

Abstract

Integrating biological imaging into early stages of the drug discovery process can provide invaluable readouts of drug activity within complex disease settings, such as cancer. Iterating this approach from initial lead compound identification in vitro to proof-of-principle in vivo analysis represents a key challenge in the drug discovery field. By embracing more complex and informative models in drug discovery, imaging can improve the fidelity and statistical robustness of preclinical cancer studies. In this Review, we highlight how combining advanced imaging with three-dimensional systems and intravital mouse models can provide more informative and disease-relevant platforms for cancer drug discovery.

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Figure 1: Applications of image-based high-content screening and intravital imaging increase the value of core elements of the drug project operating model (DPOM).
Figure 2: Engineering more predictive models for cancer research.
Figure 3: Optical imaging windows facilitate non-invasive intravital imaging of drug response in live tissue.
Figure 4: Imaging collagen abundance and fibrosis using second harmonic generation microscopy of subcutaneous patient-derived pancreatic tumours.

References

  1. Swinney, D. C. The contribution of mechanistic understanding to phenotypic screening for first-in-class medicines. J. Biomol. Screen 18, 1186–1192 (2013).

    Article  PubMed  Google Scholar 

  2. Carragher, N. O. et al. Live cell in vitro and in vivo imaging applications: accelerating drug discovery. Pharmaceutics 3, 141–170 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Timpson, P., McGhee, E. J. & Anderson, K. I. Imaging molecular dynamics in vivo—from cell biology to animal models. J. Cell Sci. 124, 2877–2890 (2011).

    Article  CAS  PubMed  Google Scholar 

  4. Paul, S. M. et al. How to improve R&D productivity: the pharmaceutical industry's grand challenge. Nature Rev. Drug Discov. 9, 203–214 (2010).

    Article  CAS  Google Scholar 

  5. Tentler, J. J. et al. Patient-derived tumour xenografts as models for oncology drug development. Nature Rev. Clin. Oncol. 9, 338–350 (2012).

    Article  CAS  Google Scholar 

  6. Kamb, A. What's wrong with our cancer models? Nature Rev. Drug Discov. 4, 161–165 (2005).

    Article  CAS  Google Scholar 

  7. Weissleder, R. & Pittet, M. J. Imaging in the era of molecular oncology. Nature 452, 580–589 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Beerling, E., Ritsma, L., Vrisekoop, N., Derksen, P. W. & van Rheenen, J. Intravital microscopy: new insights into metastasis of tumors. J. Cell Sci. 124, 299–310 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lee, J. A. & Berg, E. L. Neoclassic drug discovery: the case for lead generation using phenotypic and functional approaches. J. Biomol. Screen 18, 1143–1155 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Swinney, D. C. & Anthony, J. How were new medicines discovered? Nature Rev. Drug Discov. 10, 507–519 (2011). This paper reviews the contributions of target- and phenotypic-directed drug discovery in a retrospective analysis of all drugs approved by the FDA between 1999 and 2008.

    Article  CAS  Google Scholar 

  11. Bakal, C., Aach, J., Church, G. & Perrimon, N. Quantitative morphological signatures define local signaling networks regulating cell morphology. Science 316, 1753–1756 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Vindin, H., Bischof, L., Gunning, P. & Stehn, J. Validation of an algorithm to quantify changes in actin cytoskeletal organization. J. Biomol. Screen 19, 354–368 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Cappella, P. & Gasparri, F. Highly multiplexed phenotypic imaging for cell proliferation studies. J. Biomol. Screen 19, 145–157 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Yarrow, J. C., Totsukawa, G., Charras, G. T. & Mitchison, T. J. Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor. Chem. Biol. 12, 385–395 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Xu, G. W. et al. A high-content chemical screen identifies ellipticine as a modulator of p53 nuclear localization. Apoptosis 13, 413–422 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Young, D. W. et al. Integrating high-content screening and ligand-target prediction to identify mechanism of action. Nature Chem. Biol. 4, 59–68 (2008).

    Article  CAS  Google Scholar 

  17. Rose, R. H., Briddon, S. J. & Holliday, N. D. Bimolecular fluorescence complementation: lighting up seven transmembrane domain receptor signalling networks. Br. J. Pharmacol. 159, 738–750 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Dai, J. P. et al. Drug screening for autophagy inhibitors based on the dissociation of Beclin1-Bcl2 complex using BiFC technique and mechanism of eugenol on anti-influenza A virus activity. PLoS ONE 8, e61026 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Shinjo, S., Tashiro, E. & Imoto, M. Establishment of a new detection system for the dimerization of IRE1α by BiFC assay. Biosci. Biotechnol. Biochem. 77, 1333–1336 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Filonov, G. S. & Verkhusha, V. V. A near-infrared bifc reporter for in vivo imaging of protein-protein interactions. Chem. Biol. 20, 1078–1086 (2013). This paper describes the design of the first near-infrared BiFC reporter for in vivo protein interaction studies.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fang, D. & Kerppola, T. K. Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes. Proc. Natl Acad. Sci. USA 101, 14782–14787 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Li, R. et al. Akt SUMOylation regulates cell proliferation and tumorigenesis. Cancer Res. 73, 5742–5753 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Day, C. A., Kraft, L. J., Kang, M. & Kenworthy, A. K. Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP). Curr. Protoc. Cytom. 62, 2.19.1–2.19.29 (2012).

    Article  Google Scholar 

  24. Canel, M., Serrels, A., Anderson, K. I., Frame, M. C. & Brunton, V. G. Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane. Cell Adh. Migr. 4, 491–501 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Canel, M. et al. Quantitative in vivo imaging of the effects of inhibiting integrin signaling via Src and FAK on cancer cell movement: effects on E-cadherin dynamics. Cancer Res. 70, 9413–9422 (2010). This paper describes the combined application of intravital imaging windows with three distinct subcellular advanced techniques (photoactivation, photoswitching and FRAP) to examine tumour cell–cell adhesion strength and response to anti-invasive receptor tyrosine kinase (RTK) or endocytic drug treatment.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Serrels, A. et al. Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development. Cancer Res. 69, 2714–2719 (2009). This paper describes the first use of live in vivo FRAP to measure cell–cell junction dynamics in living solid tumour tissue: FRAP was used as a surrogate marker of the tumour dissociation response to therapeutic intervention.

    Article  CAS  PubMed  Google Scholar 

  27. Yamada, S., Pokutta, S., Drees, F., Weis, W. I. & Nelson, W. J. Deconstructing the cadherin-catenin-actin complex. Cell 123, 889–901 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. de Beco, S., Gueudry, C., Amblard, F. & Coscoy, S. Endocytosis is required for E-cadherin redistribution at mature adherens junctions. Proc. Natl Acad. Sci. USA 106, 7010–7015 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Cavey, M., Rauzi, M., Lenne, P. F. & Lecuit, T. A two-tiered mechanism for stabilization and immobilization of E-cadherin. Nature 453, 751–756 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Daddysman, M. K. & Fecko, C. J. Revisiting point FRAP to quantitatively characterize anomalous diffusion in live cells. J. Phys. Chem. B 117, 1241–1251 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Dieteren, C. E. et al. Solute diffusion is hindered in the mitochondrial matrix. Proc. Natl Acad. Sci. USA 108, 8657–8662 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Andrews, P. D. et al. Aurora B regulates MCAK at the mitotic centromere. Dev. Cell 6, 253–268 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Famulski, J. K. & Chan, G. K. Aurora B kinase-dependent recruitment of hZW10 and hROD to tensionless kinetochores. Curr. Biol. 17, 2143–2149 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Khodjakov, A. & Rieder, C. L. The sudden recruitment of γ-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules. J. Cell Biol. 146, 585–596 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Vink, M. et al. In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics. Curr. Biol. 16, 755–766 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Mueller, F., Wach, P. & McNally, J. G. Evidence for a common mode of transcription factor interaction with chromatin as revealed by improved quantitative fluorescence recovery after photobleaching. Biophys. J. 94, 3323–3339 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Mazza, D., Abernathy, A., Golob, N., Morisaki, T. & McNally, J. G. A benchmark for chromatin binding measurements in live cells. Nucleic Acids Res. 40, e119 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Misteli, T., Gunjan, A., Hock, R., Bustin, M. & Brown, D. T. Dynamic binding of histone H1 to chromatin in living cells. Nature 408, 877–881 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Kang, M., Day, C. A., DiBenedetto, E. & Kenworthy, A. K. A quantitative approach to analyze binding diffusion kinetics by confocal FRAP. Biophys. J. 99, 2737–2747 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Nouar, R., Devred, F., Breuzard, G. & Peyrot, V. FRET and FRAP imaging: approaches to characterise tau and stathmin interactions with microtubules in cells. Biol. Cell 105, 149–161 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Murthy, K. & Wadsworth, P. Dual role for microtubules in regulating cortical contractility during cytokinesis. J. Cell Sci. 121, 2350–2359 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Kraft, L. J. & Kenworthy, A. K. Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4B(C74A) in live cells using Forster resonance energy transfer and fluorescence recovery after photobleaching. J. Biomed. Opt. 17, 011008 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schneider, K. et al. Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling. Nucleic Acids Res. 41, 4860–4876 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Schermelleh, L. et al. Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation. Nucleic Acids Res. 35, 4301–4312 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Spada, F. et al. DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells. J. Cell Biol. 176, 565–571 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Agasti, S. S. et al. Dual imaging and photoactivated nanoprobe for controlled cell tracking. Small 9, 222–227 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Wang, X., He, L., Wu, Y. I., Hahn, K. M. & Montell, D. J. Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nature Cell Biol. 12, 591–597 (2010).

    Article  CAS  PubMed  Google Scholar 

  48. Yoo, S. K. et al. Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev. Cell 18, 226–236 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Frost, N. A., Lu, H. E. & Blanpied, T. A. Optimization of cell morphology measurement via single-molecule tracking PALM. PLoS ONE 7, e36751 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Roy, S., Yang, G., Tang, Y. & Scott, D. A. A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution. Nature Protoc. 7, 62–68 (2012).

    Article  CAS  Google Scholar 

  51. Caswell, P. T. et al. Rab25 associates with α5β1 integrin to promote invasive migration in 3D microenvironments. Dev. Cell 13, 496–510 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Amornphimoltham, P. et al. Rab25 regulates invasion and metastasis in head and neck cancer. Clin. Cancer Res. 19, 1375–1388 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ritsma, L. et al. Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis. Sci. Transl Med. 4, 158ra145 (2012). This paper provides an insight into the progressive nature and capacity of intravital imaging windows to monitor late stages of metastasis from deep within the body cavity at high resolution, revealing a time-dependent aspect to when anti-migratory targeting can be effective.

    Article  CAS  PubMed  Google Scholar 

  54. Kedrin, D. et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nature Methods 5, 1019–1021 (2008). This paper describes the combined application of intravital imaging windows with photoactivation for repeated imaging and tracking of tumour population dynamics in mammary tumours.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Yu, X. et al. N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods. J. Cell Biol. 199, 527–544 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Deakin, N. O., Ballestrem, C. & Turner, C. E. Paxillin and Hic-5 interaction with vinculin is differentially regulated by Rac1 and RhoA. PLoS ONE 7, e37990 (2012). This study demonstrates the application of FRET biosensors to provide novel insight into protein–protein interactions within cell adhesions and their distinction between 2D and 3D in vitro models.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Wouters, F. S., Verveer, P. J. & Bastiaens, P. I. Imaging biochemistry inside cells. Trends Cell Biol. 11, 203–211 (2001).

    Article  CAS  PubMed  Google Scholar 

  58. Fruhwirth, G. O. et al. How Forster resonance energy transfer imaging improves the understanding of protein interaction networks in cancer biology. Chemphyschem 12, 442–461 (2011). This is a comprehensive overview of the potential use of FRET-based biosensor imaging in cancer.

    Article  CAS  PubMed  Google Scholar 

  59. Seong, J. et al. Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer. Nature Commun. 2, 406 (2011).

    Article  CAS  Google Scholar 

  60. Wang, Y. et al. Visualizing the mechanical activation of Src. Nature 434, 1040–1045 (2005).

    Article  CAS  PubMed  Google Scholar 

  61. Hirata, E. et al. In vivo fluorescence resonance energy transfer imaging reveals differential activation of Rho-family GTPases in glioblastoma cell invasion. J. Cell Sci. 125, 858–868 (2012).

    Article  CAS  PubMed  Google Scholar 

  62. Ouyang, M. et al. Visualization of polarized membrane type 1 matrix metalloproteinase activity in live cells by fluorescence resonance energy transfer imaging. J. Biol. Chem. 283, 17740–17748 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Lu, S. et al. Quantitative FRET imaging to visualize the invasiveness of live breast cancer cells. PLoS ONE 8, e58569 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Ouyang, M. et al. Simultaneous visualization of protumorigenic Src and MT1-MMP activities with fluorescence resonance energy transfer. Cancer Res. 70, 2204–2212 (2010). This paper describes dual FLIM–FRET imaging of spectrally distinct biosensors for MT1MMP and SRC activity.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Luo, K. Q., Yu, V. C., Pu, Y. & Chang, D. C. Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells. Biochem. Biophys. Res. Commun. 283, 1054–1060 (2001).

    Article  CAS  PubMed  Google Scholar 

  66. Yoshizaki, H. et al. Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J. Cell Biol. 162, 223–232 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Gavet, O. & Pines, J. Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis. J. Cell Biol. 189, 247–259 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Gavet, O. & Pines, J. Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev. Cell 18, 533–543 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Nobis, M. et al. Intravital FLIM-FRET imaging reveals dasatinib-induced spatial control of Src in pancreatic cancer. Cancer Res. 75, 4674–4686 (2013). This paper highlights the usefulness of FRET-biosensor expression in target tissue, providing a reversible and dynamic readout of target inactivation and clearance response to drug treatment in live tumours.

    Article  CAS  Google Scholar 

  70. Milligan, G. Applications of bioluminescence- and fluorescence resonance energy transfer to drug discovery at G protein-coupled receptors. Eur. J. Pharm. Sci. 21, 397–405 (2004).

    Article  CAS  PubMed  Google Scholar 

  71. Tian, H., Ip, L., Luo, H., Chang, D. C. & Luo, K. Q. A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine. Br. J. Pharmacol. 150, 321–334 (2007).

    Article  CAS  PubMed  Google Scholar 

  72. Stockholm, D. et al. Imaging calpain protease activity by multiphoton FRET in living mice. J. Mol. Biol. 346, 215–222 (2005).

    Article  CAS  PubMed  Google Scholar 

  73. Janssen, A., Beerling, E., Medema, R. & van Rheenen, J. Intravital FRET imaging of tumor cell viability and mitosis during chemotherapy. PLoS ONE 8, e64029 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Matsuda, T., Horikawa, K., Saito, K. & Nagai, T. Highlighted Ca2+ imaging with a genetically encoded 'caged' indicator. Sci. Rep. 3, 1398 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Demarco, I. A., Periasamy, A., Booker, C. F. & Day, R. N. Monitoring dynamic protein interactions with photoquenching FRET. Nature Methods 3, 519–524 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Subach, F. V. et al. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET. Chem. Biol. 17, 745–755 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Peter, M. et al. Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions. Biophys. J. 88, 1224–1237 (2005).

    Article  CAS  PubMed  Google Scholar 

  78. Talbot, C. B. et al. High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis. J. Biophoton. 1, 514–521 (2008). This study provides the first example of a FLIM biosensor incorporated into a high-throughput image-based screening platform.

    Article  Google Scholar 

  79. Grecco, H. E. et al. In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays. Nature Methods 7, 467–472 (2010). This paper describes the elegant use of high-speed FLIM to measure FRET in a high-throughput setting, which provides insight into the concerted activity and network redundancy in epidermal growth factor receptor (EGFR) signalling. It is applicable to RTK-targeted drug resistance and feedback.

    Article  CAS  PubMed  Google Scholar 

  80. Kumar, S. et al. FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ. Chemphyschem 12, 609–626 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Alibhai, D. et al. Automated fluorescence lifetime imaging plate reader and its application to Forster resonant energy transfer readout of Gag protein aggregation. J. Biophoton. 6, 398–408 (2013).

    Article  CAS  Google Scholar 

  82. Grecco, H. E., Roda-Navarro, P., Fengler, S. & Bastiaens, P. I. High-throughput quantification of posttranslational modifications in situ by CA-FLIM. Methods Enzymol. 500, 37–58 (2011).

    Article  CAS  PubMed  Google Scholar 

  83. McGhee, E. J. et al. FLIM-FRET imaging in vivo reveals 3D-environment spatially regulates RhoGTPase activity during cancer cell invasion. Small GTPases 2, 239–244 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  84. Worth, D. C. & Parsons, M. Advances in imaging cell-matrix adhesions. J. Cell Sci. 123, 3629–3638 (2010).

    Article  CAS  PubMed  Google Scholar 

  85. Jares-Erijman, E. A. & Jovin, T. M. FRET imaging. Nature Biotech. 21, 1387–1395 (2003).

    Article  CAS  Google Scholar 

  86. Berney, C. & Danuser, G. FRET or no FRET: a quantitative comparison. Biophys. J. 84, 3992–4010 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Roh-Johnson, M. et al. Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation. Oncogene http://dx.doi.org/10.1038/onc.2013.377 (2013).

  88. Ottobrini, L., Martelli, C., Trabattoni, D. L., Clerici, M. & Lucignani, G. In vivo imaging of immune cell trafficking in cancer. Eur. J. Nucl. Med. Mol. Imag. 38, 949–968 (2011).

    Article  Google Scholar 

  89. Chtanova, T. et al. Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice. J. Biophoton http://dx.doi.org/10.1002/jbio.201200175 (2012).

  90. Makrogianneli, K. et al. Integrating receptor signal inputs that influence small Rho GTPase activation dynamics at the immunological synapse. Mol. Cell. Biol. 29, 2997–3006 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Lohela, M. & Werb, Z. Intravital imaging of stromal cell dynamics in tumors. Curr. Opin. Genet. Dev. 20, 72–78 (2010).

    Article  CAS  PubMed  Google Scholar 

  92. Egeblad, M., Nakasone, E. S. & Werb, Z. Tumors as organs: complex tissues that interface with the entire organism. Dev. Cell 18, 884–901 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Grant, D. M. et al. Multiplexed FRET to image multiple signaling events in live cells. Biophys. J. 95, L69–L71 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Rao, J., Bhattacharya, D., Banerjee, B., Sarin, A. & Shivashankar, G. V. Trichostatin-A induces differential changes in histone protein dynamics and expression in HeLa cells. Biochem. Biophys. Res. Commun. 363, 263–268 (2007).

    Article  CAS  PubMed  Google Scholar 

  95. Li, W., Wang, Y., Shao, H., He, Y. & Ma, H. Probing rotation dynamics of biomolecules using polarization based fluorescence microscopy. Microsc. Res. Tech. 70, 390–395 (2007).

    Article  CAS  PubMed  Google Scholar 

  96. Cao, Z., Huang, C. C. & Tan, W. Nuclease resistance of telomere-like oligonucleotides monitored in live cells by fluorescence anisotropy imaging. Anal. Chem. 78, 1478–1484 (2006).

    Article  CAS  PubMed  Google Scholar 

  97. Sharma, P. et al. Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 116, 577–589 (2004).

    Article  CAS  PubMed  Google Scholar 

  98. Matthews, D. R. et al. A multi-functional imaging approach to high-content protein interaction screening. PLoS ONE 7, e33231 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Eichorst, J. P., Clegg, R. M. & Wang, Y. Red-shifted fluorescent proteins monitor enzymatic activity in live HT-1080 cells with fluorescence lifetime imaging microscopy (FLIM). J. Microsc. 248, 77–89 (2012).

    Article  CAS  PubMed  Google Scholar 

  100. Eichorst, J. P., Huang, H., Clegg, R. M. & Wang, Y. Phase differential enhancement of FLIM to distinguish FRET components of a biosensor for monitoring molecular activity of membrane type 1 matrix metalloproteinase in live cells. J. Fluoresc 21, 1763–1777 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Tyas, L., Brophy, V. A., Pope, A., Rivett, A. J. & Tavare, J. M. Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer. EMBO Rep. 1, 266–270 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Shcherbakova, D. M., Hink, M. A., Joosen, L., Gadella, T. W. & Verkhusha, V. V. An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J. Am. Chem. Soc. 134, 7913–7923 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Keese, M., Yagublu, V., Schwenke, K., Post, S. & Bastiaens, P. Fluorescence lifetime imaging microscopy of chemotherapy-induced apoptosis resistance in a syngenic mouse tumor model. Int. J. Cancer 126, 104–113 (2010).

    Article  CAS  PubMed  Google Scholar 

  104. Tomura, M. et al. Time-lapse observation of cellular function with fluorescent probe reveals novel CTL-target cell interactions. Int. Immunol. 21, 1145–1150 (2009).

    Article  CAS  PubMed  Google Scholar 

  105. Yamaguchi, Y. et al. Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure. J. Cell Biol. 195, 1047–1060 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Ting, A. Y., Kain, K. H., Klemke, R. L. & Tsien, R. Y. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc. Natl Acad. Sci. USA 98, 15003–15008 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Kunkel, M. T., Ni, Q., Tsien, R. Y., Zhang, J. & Newton, A. C. Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter. J. Biol. Chem. 280, 5581–5587 (2005).

    Article  CAS  PubMed  Google Scholar 

  109. Sasaki, K., Sato, M. & Umezawa, Y. Fluorescent indicators for Akt/protein kinase B and dynamics of Akt activity visualized in living cells. J. Biol. Chem. 278, 30945–30951 (2003).

    Article  CAS  PubMed  Google Scholar 

  110. Yoshizaki, H., Mochizuki, N., Gotoh, Y. & Matsuda, M. Akt-PDK1 complex mediates epidermal growth factor-induced membrane protrusion through Ral activation. Mol. Biol. Cell 18, 119–128 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Mochizuki, N. et al. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411, 1065–1068 (2001).

    Article  CAS  PubMed  Google Scholar 

  112. Itoh, R. E. et al. Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells. Mol. Cell. Biol. 22, 6582–6591 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Kardash, E. et al. A role for Rho GTPases and cell-cell adhesion in single-cell motility in vivo. Nature Cell Biol. 12 (Suppl. 1–11), 47–53 (2010).

    Article  CAS  PubMed  Google Scholar 

  114. Timpson, P. et al. Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53. Cancer Res. 71, 747–757 (2011). This study demonstrates that subcellular FLIM–FRET imaging and targeting can reveal subtle but vital signalling events that drive tumour invasion in vivo.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Goto, A. et al. GDNF and endothelin 3 regulate migration of enteric neural crest-derived cells via protein kinase A and Rac1. J. Neurosci. 33, 4901–4912 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Johnsson, A. E. et al. The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues. Cell Reports 6, 1153–1164 (2014). A RAC–FRET biosensor mouse was generated, allowing the spatiotemporal activity of RAC GTPase to be assessed in primary neutrophils and multiple organ types, such as the pancreas, liver, intestine and mammary tissue, in real-time. By crossing this inducible mouse with distinct tumour mouse models, we could expand our knowledge of how RAC GTPase behaves in a native host mammalian tissue upon drug treatment.

    Article  CAS  PubMed  Google Scholar 

  117. Fehr, M., Lalonde, S., Lager, I., Wolff, M. W. & Frommer, W. B. In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J. Biol. Chem. 278, 19127–19133 (2003).

    Article  CAS  PubMed  Google Scholar 

  118. Okumoto, S. et al. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc. Natl Acad. Sci. USA 102, 8740–8745 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Imamura, H. et al. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc. Natl Acad. Sci. USA 106, 15651–15656 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  120. Ritsma, L., Vrisekoop, N. & van Rheenen, J. In vivo imaging and histochemistry are combined in the cryosection labelling and intravital microscopy technique. Nature Commun. 4, 2366 (2013). In this study, in a similar manner to electron microscopy, intravital images were correlated with cryosection labelling to provide post-imaging detail of the sample. Along with instant readouts from intravital imaging, this provides added contextual value and the long-term capacity to re-evaluate the sample after live imaging.

    Article  Google Scholar 

  121. Potzkei, J. et al. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor. BMC Biol. 10, 28 (2012). This paper describes the development of FluBO, an intramolecular FRET-based biosensor for detecting intracellular oxygen. FluBO uses an oxygen-insensitive donor fluorescent protein that is intramolecularly linked to an oxygen-sensitive acceptor fluorescent protein, and thus FRET only occurs in the presence of oxygen. This biosensor could eventually be applied to measure cellular hypoxia for in vivo cancer research.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Urra, J. et al. A genetically encoded ratiometric sensor to measure extracellular pH in microdomains bounded by basolateral membranes of epithelial cells. Pflugers Arch. 457, 233–242 (2008).

    Article  CAS  PubMed  Google Scholar 

  123. Awaji, T., Hirasawa, A., Shirakawa, H., Tsujimoto, G. & Miyazaki, S. Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains. Biochem. Biophys. Res. Commun. 289, 457–462 (2001).

    Article  CAS  PubMed  Google Scholar 

  124. Drepper, T. et al. Reporter proteins for in vivo fluorescence without oxygen. Nature Biotech. 25, 443–445 (2007).

    Article  CAS  Google Scholar 

  125. Drepper, T. et al. Flavin mononucleotide-based fluorescent reporter proteins outperform green fluorescent protein-like proteins as quantitative in vivo real-time reporters. Appl. Environ. Microbiol. 76, 5990–5994 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Piljic, A. & Schultz, C. Simultaneous recording of multiple cellular events by FRET. ACS Chem. Biol. 3, 156–160 (2008).

    Article  CAS  PubMed  Google Scholar 

  127. Peyker, A., Rocks, O. & Bastiaens, P. I. Imaging activation of two Ras isoforms simultaneously in a single cell. Chembiochem 6, 78–85 (2005).

    Article  CAS  PubMed  Google Scholar 

  128. Kamioka, Y. et al. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors. Cell Struct. Funct. 37, 65–73 (2012). This paper describes the generation of FRET biosensor mice.

    Article  CAS  PubMed  Google Scholar 

  129. Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl Acad. Sci. USA 101, 10554–10559 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Atkin, S. D. et al. Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo. J. Neurosci. Methods 181, 212–226 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Hara, M. et al. Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice. Am. J. Physiol. Cell Physiol. 287, C932–C938 (2004).

    Article  CAS  PubMed  Google Scholar 

  132. Isotani, E. et al. Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse. Proc. Natl Acad. Sci. USA 101, 6279–6284 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Bartoli, M. et al. A mouse model for monitoring calpain activity under physiological and pathological conditions. J. Biol. Chem. 281, 39672–39680 (2006).

    Article  CAS  PubMed  Google Scholar 

  134. Nikolaev, V. O., Bunemann, M., Schmitteckert, E., Lohse, M. J. & Engelhardt, S. Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined β2-adrenergic receptor-mediated signaling. Circ. Res. 99, 1084–1091 (2006).

    Article  CAS  PubMed  Google Scholar 

  135. Calebiro, D. et al. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol. 7, e1000172 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008).

    Article  CAS  PubMed  Google Scholar 

  137. Yamamoto, N. et al. Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression. Cancer Res. 64, 4251–4256 (2004).

    Article  CAS  PubMed  Google Scholar 

  138. Yamamoto, N. et al. Determination of clonality of metastasis by cell-specific color-coded fluorescent-protein imaging. Cancer Res. 63, 7785–7790 (2003).

    CAS  PubMed  Google Scholar 

  139. Hoffman, R. M. The multiple uses of fluorescent proteins to visualize cancer in vivo. Nature Rev. Cancer 5, 796–806 (2005).

    Article  CAS  Google Scholar 

  140. Day, R. N. & Davidson, M. W. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells. Bioessays 34, 341–350 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Newman, R. H., Fosbrink, M. D. & Zhang, J. Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem. Rev. 111, 3614–3666 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Sahai, E. & Marshall, C. J. Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nature Cell Biol. 5, 711–719 (2003).

    Article  CAS  PubMed  Google Scholar 

  143. Friedl, P. & Wolf, K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nature Rev. Cancer 3, 362–374 (2003).

    Article  CAS  Google Scholar 

  144. Friedl, P., Sahai, E., Weiss, S. & Yamada, K. M. New dimensions in cell migration. Nature Rev. Mol. Cell. Biol. 13, 743–747 (2012). This paper gives a comprehensive insight into the appropriate use of 3D matrices to mimic in vivo homestasis or disease conditions.

    Article  CAS  Google Scholar 

  145. Spence, H. J., Timpson, P., Tang, H. R., Insall, R. H. & Machesky, L. M. Scar/WAVE3 contributes to motility and plasticity of lamellipodial dynamics but not invasion in three dimensions. Biochem. J. 448, 35–42 (2012).

    Article  CAS  PubMed  Google Scholar 

  146. Wittig, R. et al. Biosensor-expressing spheroid cultures for imaging of drug-induced effects in three dimensions. J. Biomol. Screen 18, 736–743 (2013).

    Article  CAS  PubMed  Google Scholar 

  147. le Roux, L. et al. Optimizing imaging of three-dimensional multicellular tumor spheroids with fluorescent reporter proteins using confocal microscopy. Mol. Imag. 7, 214–221 (2008).

    Article  Google Scholar 

  148. Uchugonova, A. et al. Multiphoton tomography visualizes collagen fibers in the tumor microenvironment that maintain cancer-cell anchorage and shape. J. Cell Biochem. 114, 99–102 (2013).

    Article  CAS  PubMed  Google Scholar 

  149. Pickl, M. & Ries, C. H. Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab. Oncogene 28, 461–468 (2009).

    Article  CAS  PubMed  Google Scholar 

  150. Calvo, F. et al. Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nature Cell Biol. 15, 637–646 (2013).

    Article  CAS  PubMed  Google Scholar 

  151. Nurmenniemi, S. et al. A novel organotypic model mimics the tumor microenvironment. Am. J. Pathol. 175, 1281–1291 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Gaggioli, C. et al. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nature Cell Biol. 9, 1392–1400 (2007). This paper presents a strong argument for the use of organotypic co-culture models to investigate the complex systems involved in cancer metastasis.

    Article  CAS  PubMed  Google Scholar 

  153. Rothberg, J. M., Sameni, M., Moin, K. & Sloane, B. F. Live-cell imaging of tumor proteolysis: impact of cellular and non-cellular microenvironment. Biochim. Biophys. Acta 1824, 123–132 (2012).

    Article  CAS  PubMed  Google Scholar 

  154. Wang, R. et al. Three-dimensional co-culture models to study prostate cancer growth, progression, and metastasis to bone. Semin. Cancer Biol. 15, 353–364 (2005).

    Article  CAS  PubMed  Google Scholar 

  155. Talukdar, S. & Kundu, S. C. A. non-mulberry silk fibroin protein based 3D in vitro tumor model for evaluation of anticancer drug activity. Adv. Funct. Mater. 22, 4778–4788 (2012).

    Article  CAS  Google Scholar 

  156. Serebriiskii, I., Castello-Cros, R., Lamb, A., Golemis, E. A. & Cukierman, E. Fibroblast-derived 3D matrix differentially regulates the growth and drug-responsiveness of human cancer cells. Matrix Biol. 27, 573–585 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. Loessner, D. et al. Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells. Biomaterials 31, 8494–8506 (2010).

    Article  CAS  PubMed  Google Scholar 

  158. Schrader, J. et al. Matrix stiffness modulates proliferation, chemotherapeutic response, and dormancy in hepatocellular carcinoma cells. Hepatology 53, 1192–1205 (2011).

    Article  CAS  PubMed  Google Scholar 

  159. Sethi, T. et al. Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo. Nature Med. 5, 662–668 (1999).

    Article  CAS  PubMed  Google Scholar 

  160. Longati, P. et al. 3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing. BMC Cancer 13, 95 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. Straussman, R. et al. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487, 500–504 (2012). This study demonstrated that stromal cells conferred innate chemoresistance to cancer cells through treatment with an array of 35 anticancer drugs in 45 cancer cells, cultured alone or in co-culture with stromal cells.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  162. Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139, 891–906 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  163. Mih, J. D., Marinkovic, A., Liu, F., Sharif, A. S. & Tschumperlin, D. J. Matrix stiffness reverses the effect of actomyosin tension on cell proliferation. J. Cell Sci. 125, 5974–5983 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  164. Zustiak, S., Nossal, R. & Sackett, D. L. Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs. Biotechnol. Bioeng. 111, 396–403 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  165. Wolf, K. et al. Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nature Cell Biol. 9, 893–904 (2007).

    Article  CAS  PubMed  Google Scholar 

  166. Tozluoglu, M. et al. Matrix geometry determines optimal cancer cell migration strategy and modulates response to interventions. Nature Cell Biol. 15, 751–762 (2013).

    Article  CAS  PubMed  Google Scholar 

  167. Boghaert, E. et al. Host epithelial geometry regulates breast cancer cell invasiveness. Proc. Natl Acad. Sci. USA 109, 19632–19637 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  168. Radisky, D. C. & Nelson, C. M. Regulation of mechanical stress by mammary epithelial tissue structure controls breast cancer cell invasion. Oncotarget 4, 498–499 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  169. Jacobetz, M. A. et al. Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer. Gut 62, 112–120 (2013).

    Article  CAS  PubMed  Google Scholar 

  170. Olive, K. P. et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324, 1457–1461 (2009). In this study, SHG imaging reveals that targeting the tumour stromal compartment (ECM) before drug treatment can enhance drug delivery and improve disease outcome. This study led to a shift in the field of dual-targeted therapy in pancreatic cancer.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  171. Provenzano, P. P. et al. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell 21, 418–429 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Yu, M. & Tannock, I. F. Targeting tumor architecture to favor drug penetration: a new weapon to combat chemoresistance in pancreatic cancer? Cancer Cell 21, 327–329 (2012). References 169 and 172 reveal that alterations in ECM integrity can influence drug targeting via reduced ECM content and improved vascularity or drug delivery.

    Article  CAS  PubMed  Google Scholar 

  173. Raub, C. B., Putnam, A. J., Tromberg, B. J. & George, S. C. Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy. Acta Biomater. 6, 4657–4665 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  174. Samuel, M. S. et al. Actomyosin-mediated cellular tension drives increased tissue stiffness and β-catenin activation to induce epidermal hyperplasia and tumor growth. Cancer Cell 19, 776–791 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  175. Cicchi, R. et al. Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy. J. Biophoton. 3, 34–43 (2010).

    Article  CAS  Google Scholar 

  176. Bakker, G. J., Andresen, V., Hoffman, R. M. & Friedl, P. Fluorescence lifetime microscopy of tumor cell invasion, drug delivery, and cytotoxicity. Methods Enzymol. 504, 109–125 (2012). This paper highlights the feasibility of high-speed FLIM–FRET in 3D organotypic or complex settings, allowing cancer cell invasion, apoptosis and drug uptake to be quantified with subcellular resolution.

    Article  CAS  PubMed  Google Scholar 

  177. Bremer, C., Tung, C. H. & Weissleder, R. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nature Med. 7, 743–748 (2001).

    Article  CAS  PubMed  Google Scholar 

  178. Contag, P. R. Whole-animal cellular and molecular imaging to accelerate drug development. Drug Discov. Today 7, 555–562 (2002).

    Article  CAS  PubMed  Google Scholar 

  179. Graves, E. E., Weissleder, R. & Ntziachristos, V. Fluorescence molecular imaging of small animal tumor models. Curr. Mol. Med. 4, 419–430 (2004).

    Article  CAS  PubMed  Google Scholar 

  180. Hoffman, R. M. & Yang, M. Whole-body imaging with fluorescent proteins. Nature Protoc. 1, 1429–1438 (2006).

    Article  CAS  Google Scholar 

  181. Hayashi, K. et al. Real-time imaging of tumor-cell shedding and trafficking in lymphatic channels. Cancer Res. 67, 8223–8228 (2007).

    Article  CAS  PubMed  Google Scholar 

  182. Yamauchi, K. et al. Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system. Cancer Res. 66, 4208–4214 (2006).

    Article  CAS  PubMed  Google Scholar 

  183. Yang, M. et al. Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model. Proc. Natl Acad. Sci. USA 99, 3824–3829 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  184. Giampieri, S. et al. Localized and reversible TGFβ signalling switches breast cancer cells from cohesive to single cell motility. Nature Cell Biol. 11, 1287–1296 (2009).

    Article  CAS  PubMed  Google Scholar 

  185. Li, A. et al. Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod- driven motility and cell-cycle progression. Dev. Cell 21, 722–734 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  186. Ladhani, O., Sanchez-Martinez, C., Orgaz, J. L., Jimenez, B. & Volpert, O. V. Pigment epithelium-derived factor blocks tumor extravasation by suppressing amoeboid morphology and mesenchymal proteolysis. Neoplasia 13, 633–642 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  187. 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, e25294 (2013).

    Article  PubMed  Google Scholar 

  188. Ahmed, F. et al. GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice. Cancer Res. 62, 7166–7169 (2002).

    CAS  PubMed  Google Scholar 

  189. Zomer, A. et al. Intravital imaging of cancer stem cell plasticity in mammary tumors. Stem Cells 31, 602–606 (2013). This paper describes the elegant use of simultaneous cell tagging to track cell fate and clonal progeny, and this approach was subsequently used in combination with imaging windows to monitor cell fate in tumour tissue.

    Article  CAS  PubMed  Google Scholar 

  190. Barretto, R. P. et al. Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy. Nature Med. 17, 223–228 (2011).

    Article  CAS  PubMed  Google Scholar 

  191. Tang, J. C. et al. A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation. Cell 154, 928–939 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  192. Momiyama, M. et al. Imaging the efficacy of UVC irradiation on superficial brain tumors and metastasis in live mice at the subcellular level. J. Cell Biochem. 114, 428–434 (2013).

    Article  CAS  PubMed  Google Scholar 

  193. Morton, J. P. et al. Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. Proc. Natl Acad. Sci. USA 107, 246–251 (2010). This paper describes how whole-body imaging and single cell tagging revealed a role for p53 in outgrowth of senescence, which resulted in tumour progression.

    Article  PubMed  Google Scholar 

  194. Morton, J. P. et al. Dasatinib inhibits the development of metastases in a mouse model of pancreatic ductal adenocarcinoma. Gastroenterology 139, 292–303 (2010).

    Article  CAS  PubMed  Google Scholar 

  195. Ahmad, I. et al. β-Catenin activation synergizes with PTEN loss to cause bladder cancer formation. Oncogene 30, 178–189 (2011).

    Article  CAS  PubMed  Google Scholar 

  196. Cole, A. M. et al. p21 loss blocks senescence following Apc loss and provokes tumourigenesis in the renal but not the intestinal epithelium. EMBO Mol. Med. 2, 472–486 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  197. Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    Article  CAS  PubMed  Google Scholar 

  198. Roth, S. et al. Paneth cells in intestinal homeostasis and tissue injury. PLoS ONE 7, e38965 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  199. Myant, K. B. et al. ROS production and NF-κB activation triggered by RAC1 facilitate WNT-driven intestinal stem cell proliferation and colorectal cancer initiation. Cell Stem Cell 12, 761–773 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  200. Chan, K. T. et al. Intravital imaging of a spheroid-based orthotopic model of melanoma in the mouse ear skin. IntraVital 2, e25805 (2013).

    Article  PubMed  Google Scholar 

  201. Lindsay, C. R. et al. P-Rex1 is required for efficient melanoblast migration and melanoma metastasis. Nature Commun. 2, 555 (2011).

    Article  CAS  Google Scholar 

  202. Doyle, B. et al. p53 mutation and loss have different effects on tumourigenesis in a novel mouse model of pleomorphic rhabdomyosarcoma. J. Pathol. 222, 129–137 (2010).

    Article  CAS  PubMed  Google Scholar 

  203. Gritsenko, P. G., Ilina, O. & Friedl, P. Interstitial guidance of cancer invasion. J. Pathol. 226, 185–199 (2012).

    Article  CAS  PubMed  Google Scholar 

  204. Dirat, B. et al. Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion. Cancer Res. 71, 2455–2465 (2011).

    Article  CAS  PubMed  Google Scholar 

  205. Nieman, K. M. et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nature Med. 17, 1498–1503 (2011).

    Article  CAS  PubMed  Google Scholar 

  206. Wolf, K. et al. Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force. J. Cell Biol. 201, 1069–1084 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  207. Le Devedec, S. E. et al. Two-photon intravital multicolour imaging to study metastatic behaviour of cancer cells in vivo. Methods Mol. Biol. 769, 331–349 (2011).

    Article  CAS  PubMed  Google Scholar 

  208. Coffey, S. E., Giedt, R. J. & Weissleder, R. Automated analysis of clonal cancer cells by intravital imaging. IntraVital 2, e26138 (2013).

    Article  Google Scholar 

  209. Wyckoff, J. et al. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res. 64, 7022–7029 (2004).

    Article  CAS  PubMed  Google Scholar 

  210. Wyckoff, J. B. et al. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res. 67, 2649–2656 (2007).

    Article  CAS  PubMed  Google Scholar 

  211. Lin, E. Y. et al. Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res. 66, 11238–11246 (2006).

    Article  CAS  PubMed  Google Scholar 

  212. Zhou, Z. N. et al. Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo. Oncogene http://dx.doi.org/10.1038/onc.2013.363 (2013).

  213. Xu, Z. et al. Role of pancreatic stellate cells in pancreatic cancer metastasis. Am. J. Pathol. 177, 2585–2596 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  214. Brown, E. et al. Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nature Med. 9, 796–800 (2003).

    Article  CAS  PubMed  Google Scholar 

  215. Kienast, Y. et al. Real-time imaging reveals the single steps of brain metastasis formation. Nature Med. 16, 116–122 (2010). This study demonstrates the use of CIW technology to track metastatic site colonization of cancer cells.

    Article  CAS  PubMed  Google Scholar 

  216. Gligorijevic, B. & Condeelis, J. Stretching the timescale of intravital imaging in tumors. Cell Adh Migr. 3, 313–315 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  217. Gligorijevic, B., Kedrin, D., Segall, J. E., Condeelis, J. & van Rheenen, J. Dendra2 photoswitching through the mammary imaging window. J. Vis. Exp. 28, 1278 (2009).

    Google Scholar 

  218. Ritsma, L. et al. Surgical implantation of an abdominal imaging window for intravital microscopy. Nature Protoc. 8, 583–594 (2013). This paper describes the application of AIWs to repeatedly and non-invasively monitor organs from deep within the abdominal cavity. Liver, pancreas, intestine and kidney were imaged at high resolution in situ.

    Article  CAS  Google Scholar 

  219. Giedt, R. J., Koch, P. D. & Weissleder, R. Single cell analysis of drug distribution by intravital imaging. PLoS ONE 8, e60988 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  220. Agasti, S. S., Laughney, A. M., Kohler, R. H. & Weissleder, R. A photoactivatable drug-caged fluorophore conjugate allows direct quantification of intracellular drug transport. Chem. Commun. 49, 11050–11052 (2013).

    Article  CAS  Google Scholar 

  221. Thurber, G. M. et al. Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo. Nature Commun. 4, 1504 (2013). In this study, the authors use intravital imaging to monitor a PARP1 inhibitor reaching its target compartment within the cell in vivo.

    Article  CAS  Google Scholar 

  222. US Department of Health and Human Services. Food and Drug Administration (FDA) Centre for Drug Evaluation and Research (CDER). Guidance for Industry: Codevelopment of two or more new investigational drugs for use in combination. June 2013. Clinical Medicine.

  223. Hughes, B. Novel agents combined get own guidance. Nature Biotech. 29, 174 (2011).

    Article  CAS  Google Scholar 

  224. Natale, D., Soriano, S. F., Coelho, F. M., Hons, M. & Stein, J. V. Comprehensive assessment of quantum dots for multispectral twophoton imaging of dynamic leukocyte migration in lymph nodes. IntraVital 2, e25745 (2013).

    Article  Google Scholar 

  225. Suetsugu, A. et al. Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude-mouse models. Adv. Drug Deliv. Rev. 65, 383–390 (2013).

    Article  CAS  PubMed  Google Scholar 

  226. Chaudhry, S. I. et al. Autocrine IL-1β-TRAF6 signalling promotes squamous cell carcinoma invasion through paracrine TNFα signalling to carcinoma-associated fibroblasts. Oncogene 32, 747–758 (2013).

    Article  CAS  PubMed  Google Scholar 

  227. Zhang, J. & Liu, J. Tumor stroma as targets for cancer therapy. Pharmacol. Ther. 137, 200–215 (2013).

    Article  CAS  PubMed  Google Scholar 

  228. Bousso, P. & Moreau, H. D. Functional immunoimaging: the revolution continues. Nature Rev. Immunol. 12, 858–864 (2012).

    Article  CAS  Google Scholar 

  229. Carmeliet, P. & Jain, R. K. Molecular mechanisms and clinical applications of angiogenesis. Nature 473, 298–307 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  230. Mellman, I., Coukos, G. & Dranoff, G. Cancer immunotherapy comes of age. Nature 480, 480–489 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  231. Clarke, J. M. & Hurwitz, H. I. Targeted inhibition of VEGF receptor 2: an update on ramucirumab. Expert Opin. Biol. Ther. 13, 1187–1196 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  232. Di Marco, M., Macchini, M., Vecchiarelli, S., Sina, S. & Biasco, G. Hedgehog signaling: from the cuirass to the heart of pancreatic cancer. Pancreatology 12, 388–393 (2012).

    Article  CAS  PubMed  Google Scholar 

  233. Hoffman, R. M. & Yang, M. Color-coded fluorescence imaging of tumor-host interactions. Nature Protoc. 1, 928–935 (2006).

    Article  CAS  Google Scholar 

  234. Moran, A. E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  235. Amoh, Y., Li, L., Katsuoka, K., Bouvet, M. & Hoffman, R. M. GFP-expressing vascularization of Gelfoam as a rapid in vivo assay of angiogenesis stimulators and inhibitors. Biotechniques 42, 294–298 (2007).

    Article  CAS  PubMed  Google Scholar 

  236. Tanaka, K. et al. In vivo real-time imaging of chemotherapy response on the liver metastatic tumor microenvironment using multiphoton microscopy. Oncol. Rep. 28, 1822–1830 (2012).

    Article  CAS  PubMed  Google Scholar 

  237. Manning, C. S. et al. Intravital imaging reveals conversion between distinct tumor vascular morphologies and localized vascular response to Sunitinib. IntraVital 2, e24790 (2013).

    Article  Google Scholar 

  238. Olivier, N. et al. Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy. Science 329, 967–971 (2010).

    Article  CAS  PubMed  Google Scholar 

  239. Weigelin, B., Bakker, G. & Friedl, P. Intravital third harmonic generation microscopy of collective melanoma cell invasion: Principles of interface guidance and microvesicle dynamics. IntraVital 1, 32–43 (2012).

    Article  PubMed  Google Scholar 

  240. Peinado, H. et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nature Med. 18, 883–891 (2012).

    Article  CAS  PubMed  Google Scholar 

  241. Tadrous, P. J. et al. Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer. J. Pathol. 199, 309–317 (2003).

    Article  PubMed  Google Scholar 

  242. Provenzano, P. P., Eliceiri, K. W. & Keely, P. J. Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment. Clin. Exp. Metastasis 26, 357–370 (2009).

    Article  CAS  PubMed  Google Scholar 

  243. McGinty, J. et al. Wide-field fluorescence lifetime imaging of cancer. Biomed. Opt. Express 1, 627–640 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  244. Estrella, V. et al. Acidity generated by the tumor microenvironment drives local invasion. Cancer Res. 73, 1524–1535 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  245. Skala, M. C. et al. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc. Natl Acad. Sci. USA 104, 19494–19499 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  246. Lecoq, J. et al. Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels. Nature Med. 17, 893–898 (2011).

    Article  CAS  PubMed  Google Scholar 

  247. Belousov, V. V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nature Methods 3, 281–286 (2006).

    Article  CAS  PubMed  Google Scholar 

  248. Parpaleix, A., Houssen, Y. G. & Charpak, S. Imaging local neuronal activity by monitoring PO2 transients in capillaries. Nature Med. 19, 241–246 (2013).

    Article  CAS  PubMed  Google Scholar 

  249. Timpson, P. et al. Organotypic collagen I assay: a malleable platform to assess cell behaviour in a 3-dimensional context. J. Vis. Exp. 56, e3089 (2011).

    Google Scholar 

  250. Thoma, C. R. et al. A high-throughput-compatible 3D microtissue co-culture system for phenotypic RNAi screening applications. J. Biomol. Screen 18, 1330–1337 (2013).

    Article  CAS  PubMed  Google Scholar 

  251. Tung, Y. C. et al. High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst 136, 473–478 (2011).

    Article  CAS  PubMed  Google Scholar 

  252. Drewitz, M. et al. Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues. Biotechnol. J. 6, 1488–1496 (2011).

    Article  CAS  PubMed  Google Scholar 

  253. Burgstaller, G., Oehrle, B., Koch, I., Lindner, M. & Eickelberg, O. Multiplex profiling of cellular invasion in 3D cell culture models. PLoS ONE 8, e63121 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  254. Truong, H. H. et al. Automated microinjection of cell-polymer suspensions in 3D ECM scaffolds for high-throughput quantitative cancer invasion screens. Biomaterials 33, 181–188 (2012).

    Article  CAS  PubMed  Google Scholar 

  255. Zervantonakis, I. K. et al. Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. Proc. Natl Acad. Sci. USA 109, 13515–13520 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  256. Echeverria, V. et al. An automated high-content assay for tumor cell migration through 3-dimensional matrices. J. Biomol. Screen 15, 1144–1151 (2010).

    Article  CAS  PubMed  Google Scholar 

  257. Kim, J. et al. A programmable microfluidic cell array for combinatorial drug screening. Lab. Chip 12, 1813–1822 (2012).

    Article  CAS  PubMed  Google Scholar 

  258. Alencar, H., Mahmood, U., Kawano, Y., Hirata, T. & Weissleder, R. Novel multiwavelength microscopic scanner for mouse imaging. Neoplasia 7, 977–983 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  259. Al-Gubory, K. H. & Houdebine, L. M. In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy. Eur. J. Cell Biol. 85, 837–845 (2006).

    Article  CAS  PubMed  Google Scholar 

  260. Kennedy, G. T. et al. A fluorescence lifetime imaging scanning confocal endomicroscope. J. Biophoton. 3, 103–107 (2010).

    Article  CAS  Google Scholar 

  261. Kiesslich, R. et al. Identification of epithelial gaps in human small and large intestine by confocal endomicroscopy. Gastroenterology 133, 1769–1778 (2007).

    Article  PubMed  Google Scholar 

  262. Stallmach, A., Schmidt, C., Watson, A. & Kiesslich, R. An unmet medical need: advances in endoscopic imaging of colorectal neoplasia. J. Biophoton. 4, 482–489 (2011).

    Article  Google Scholar 

  263. Dancik, Y., Favre, A., Loy, C. J., Zvyagin, A. V. & Roberts, M. S. Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo. J. Biomed. Opt. 18, 26022 (2013).

    Article  CAS  PubMed  Google Scholar 

  264. Leite-Silva, V. R. et al. The effect of formulation on the penetration of coated and uncoated zinc oxide nanoparticles into the viable epidermis of human skin in vivo. Eur. J. Pharm. Biopharm. 84, 297–308 (2013).

    Article  CAS  PubMed  Google Scholar 

  265. Sanchez, W. Y., Obispo, C., Ryan, E., Grice, J. E. & Roberts, M. S. Changes in the redox state and endogenous fluorescence of in vivo human skin due to intrinsic and photo-aging, measured by multiphoton tomography with fluorescence lifetime imaging. J. Biomed. Opt. 18, 061217 (2013).

    Article  PubMed  Google Scholar 

  266. Carragher, N. O. & Frame, M. C. Modelling distinct modes of tumour invasion and metastasis. Drug Discov. Today Dis. Models 8, 103–112 (2011).

    Article  CAS  Google Scholar 

  267. Sameni, M. et al. Imaging and quantifying the dynamics of tumor-associated proteolysis. Clin. Exp. Metastasis 26, 299–309 (2009).

    Article  CAS  PubMed  Google Scholar 

  268. Carragher, N. O. Profiling distinct mechanisms of tumour invasion for drug discovery: imaging adhesion, signalling and matrix turnover. Clin. Exp. Metastasis 26, 381–397 (2009).

    Article  CAS  PubMed  Google Scholar 

  269. Boimel, P. J. et al. Contribution of CXCL12 secretion to invasion of breast cancer cells. Breast Cancer Res. 14, R23 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  270. Xue, C. et al. Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. Cancer Res. 66, 192–197 (2006).

    Article  CAS  PubMed  Google Scholar 

  271. Ai, H. W., Henderson, J. N., Remington, S. J. & Campbell, R. E. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem. J. 400, 531–540 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  272. Markwardt, M. L. et al. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PLoS ONE 6, e17896 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  273. Goedhart, J. et al. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nature Methods 7, 137–139 (2010).

    Article  CAS  PubMed  Google Scholar 

  274. Goedhart, J. et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nature Commun. 3, 751 (2012).

    Article  CAS  Google Scholar 

  275. Goedhart, J., Vermeer, J. E., Adjobo-Hermans, M. J., van Weeren, L. & Gadella, T. W. Jr. Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples. PLoS ONE 2, e1011 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  276. Aoki, K., Kamioka, Y. & Matsuda, M. Fluorescence resonance energy transfer imaging of cell signaling from in vitro to in vivo: basis of biosensor construction, live imaging, and image processing. Dev. Growth Differ. 55, 515–522 (2013).

    Article  CAS  PubMed  Google Scholar 

  277. Truong, K. et al. FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule. Nature Struct. Biol. 8, 1069–1073 (2001).

    Article  CAS  PubMed  Google Scholar 

  278. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    Article  CAS  PubMed  Google Scholar 

  279. Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R. Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl Acad. Sci. USA 96, 2135–2140 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  280. Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. & Tsien, R. Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).

    Article  CAS  PubMed  Google Scholar 

  281. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotech. 20, 87–90 (2002).

    Article  CAS  Google Scholar 

  282. Evanko, D. S. & Haydon, P. G. Elimination of environmental sensitivity in a cameleon FRET-based calcium sensor via replacement of the acceptor with Venus. Cell Calcium 37, 341–348 (2005).

    Article  CAS  PubMed  Google Scholar 

  283. Vinkenborg, J. L., Evers, T. H., Reulen, S. W., Meijer, E. W. & Merkx, M. Enhanced sensitivity of FRET-based protease sensors by redesign of the GFP dimerization interface. Chembiochem 8, 1119–1121 (2007).

    Article  CAS  PubMed  Google Scholar 

  284. Ohashi, T., Galiacy, S. D., Briscoe, G. & Erickson, H. P. An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci. 16, 1429–1438 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  285. Nguyen, A. W. & Daugherty, P. S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nature Biotech. 23, 355–360 (2005).

    Article  CAS  Google Scholar 

  286. Lam, A. J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nature Methods 9, 1005–1012 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  287. Fritz, R. D. et al. A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space. Sci Signal 6, rs12 (2013).

    Article  CAS  PubMed  Google Scholar 

  288. Golynskiy, M. V., Rurup, W. F. & Merkx, M. Antibody detection by using a FRET-based protein conformational switch. Chembiochem 11, 2264–2267 (2010).

    Article  CAS  PubMed  Google Scholar 

  289. Rehm, M. et al. Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. Role of caspase-3. J. Biol. Chem. 277, 24506–24514 (2002).

    Article  CAS  PubMed  Google Scholar 

  290. Takemoto, K., Nagai, T., Miyawaki, A. & Miura, M. Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects. J. Cell Biol. 160, 235–243 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  291. Onuki, R. et al. Confirmation by FRET in individual living cells of the absence of significant amyloid β-mediated caspase 8 activation. Proc. Natl Acad. Sci. USA 99, 14716–14721 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  292. Li, M., Chen, X., Ye, Q. Z., Vogt, A. & Yin, X. M. A high-throughput FRET-based assay for determination of Atg4 activity. Autophagy 8, 401–412 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  293. Macurek, L. et al. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature 455, 119–123 (2008).

    Article  CAS  PubMed  Google Scholar 

  294. Harvey, C. D. et al. A genetically encoded fluorescent sensor of ERK activity. Proc. Natl Acad. Sci. USA 105, 19264–19269 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  295. Mizutani, T. et al. A novel FRET-based biosensor for the measurement of BCR-ABL activity and its response to drugs in living cells. Clin. Cancer Res. 16, 3964–3975 (2010).

    Article  CAS  PubMed  Google Scholar 

  296. Randriamampita, C. et al. A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse. PLoS ONE 3, e1521 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank H. Bennett, D. Herrmann, A. Magenau, A. Burgess, M. Pajic, B. Browne and C. Vennin. This work was supported by the Australian Research Council (ARC), Cancer Institute New South Wales (CINSW) and National Health and Medical Research Council (NHMRC) funding. N.O.C. is supported by a Research Councils United Kingdom (RCUK) fellowship.

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Glossary

Phenotypic screening

Assay systems that enable quantifiable measurements of cell phenotype or function that can be used to guide compound selection or iterative chemical design, often in the absence of any prior knowledge of an intended drug target.

First-in-class small molecule medicines

Newly approved medicines that have novel mechanisms of action, distinct from anything else on the market.

Target-directed drug discovery

A contemporary strategy for the identification and optimization of lead molecules and candidate drugs based on achieving high levels of potency and specificity against a nominated target that is implicated in disease progression.

Anisotropy

The anisotropy of a molecule is assessed through the simultaneous measurement of orthogonally polarized fluorescence relative to the polarization of the excitation light. Factors that determine the degree of anisotropy are protein mobility and molecular orientation. As a consequence, anisotropy can be used as a powerful and sensitive readout for binding and screening assays of protein behaviour and interactions.

Extracellular matrix

(ECM). A reinforced composite of structural proteins that is primarily composed of collagen and tissue-specific inclusions (for example, fibronectin and laminin), as well as other metabolites secreted by cells. The ECM provides structural support and biochemical signals for multicellular tissue and organ systems.

Intravital imaging windows

Windows that are surgically implanted in a mouse to allow repeated, non-invasive imaging over a long time course.

Organotypic 3D collagen I matrix

Fibroblast-driven contraction of acid-extracted collagen I is used to produce matrices with high in vivo fidelity for analysis of cell behaviour in a live in vitro setting.

Multiphoton intravital microscopy

This method reduces interference from the background by using more than one photon as a multiple of the excitation wavelength of the sample, effectively restricting interactions to the focal plane and allowing deep imaging within live tissue.

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Conway, J., Carragher, N. & Timpson, P. Developments in preclinical cancer imaging: innovating the discovery of therapeutics. Nat Rev Cancer 14, 314–328 (2014). https://doi.org/10.1038/nrc3724

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