Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors

doi:10.1016/j.ymeth.2017.04.014

Methods

Volume 128, 1 September 2017, Pages 78-94

https://doi.org/10.1016/j.ymeth.2017.04.014 Get rights and content

Highlights

•
A guide for selection of a fluorescent reporter for intravital microscopy.
•
Demonstration of adenoviral delivery of an intramolecular FRET biosensor.
•
A full protocol for an intravital skin flap imaging experiment in a xenograft.
•
Detailed protocols for FLIM-FRET imaging and analysis by intravital microscopy.
•
Microenvironmental imaging is demonstrated using oxygen-sensitive nanoparticles.

Abstract

Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug activity is assessed. Microenvironmental factors, such as hypoxia, pH, fibrosis, immune infiltration and stromal interactions have all been shown to have pronounced effects on drug activity in a more complex setting, which is often lost in simpler two- or three-dimensional assays. Here we present a practical guide for the application of intravital microscopy, looking at the available fluorescent reporters and their respective expression systems and analysis considerations. Moving in vivo, we also discuss the microscopy set up and methods available for overlaying microenvironmental context to the experimental readouts. This enables a smooth transition into applying higher fidelity intravital imaging to improve the drug discovery process.

Introduction

The ability to monitor cellular processes in real time has proved revolutionary. Since the discovery of green fluorescent protein (GFP), innumerable variants have been applied to track macroscopic growth, subcellular organelles and even single-molecules. The advent of intravital microscopy (IVM) increases the biological fidelity of such imaging, without the need to infer from a two- or three-dimensional (2D/3D) scenario. Here we will discuss the various considerations required to move research into this more physiologically relevant setting.

One of the most common uses of fluorescent proteins (FPs) is for tagging a protein of interest, where spatial information is then informed by fluorescence microscopy. However, this spatial information alone is often insufficient for drug validation studies, where details about protein-protein interactions and activity are necessary to demonstrate drug response [1], [2]. An increasingly common technique for high-throughput protein-protein interaction studies is bimolecular fluorescence complementation (BiFC), whereby two non-fluorescent halves of a FP re-associate upon interaction, forming a complete fluorescent molecule [3], [4], [5]. However, BiFC is less common in vivo [6], with more studies favoring dynamic fluorescence techniques, such as Förster resonance energy transfer (FRET), where a transient interaction between two FPs is measured, or fluorescence recovery after photobleaching (FRAP), where a powerful laser pulse is applied and recovery of fluorescence into the bleached region is monitored.

Live monitoring by these techniques using IVM can allow a more faithful readout of drug response, compared to standard 2D or 3D techniques. However, this increase in authenticity also requires some additional considerations. There is a well-established link, for instance, with tumor hypoxia and increased chemoresistance, radioresistance and metastasis [7], [8], [9]. Similarly, the burden of a dense, fibrotic extracellular matrix (ECM) is known to hamper response to therapeutics [10], [11], [12], [13], while stromal and immune interactions with the tumor can similarly favor growth [14], [15], [16], [17], [18], [19]. This microenvironmental context for drug validation studies is a necessary addition, as it is a lack of such context that is contributing to the increasing attrition rates of lead compounds in the pharmaceutical industry [20], [21], [22], [23]. Here we will demonstrate IVM techniques that facilitate both live monitoring of therapeutic response and provide microenvironmental context for this data, ultimately improving the likelihood of success in later clinical trials.

Section snippets

Using fluorescent reporters to monitor therapeutic response

The first consideration for assessing therapeutic response by IVM is the live readout. Many fluorescent reporters exist for this purpose.

Considerations for IVM of live tumor xenografts

Once you have the appropriate reporter and expression system, the next step is to progress to in vivo imaging.

IVM equipment and analysis

Intravital imaging often requires specific microscopy setups, which are covered in detail by others [28], [147]. Here, we discuss specific requirements for the use of fluorescent reporters and biosensors for IVM.

Microenvironmental context for intravital imaging

Progressing from IVM of a fluorescent reporter to additional microenvironmental readouts can be a fairly straightforward process. It is important to note however, that many fluorophores have significantly broader excitation spectra under MP excitation, compared to single-photon excitation. While this can be an advantage, allowing the excitation of several probes simultaneously, it is therefore desirable to select reporters with distinct emission wavelengths to allow robust signal separation.

Conclusions

To address increasing attrition rates in the later stages of the drug development pipeline, more faithful systems for assessing therapeutic response are necessary. Here we present a practical guide for IVM in the context of the tumor microenvironment. The first consideration is to the fluorescent reporter and it’s respective expression system and analysis methodology. Once a stable system is established, IVM of a skin flap xenograft tumor is demonstrated. While autochthonous transgenic mouse

Adenoviral FLIM-FRET imaging

For Eevee-Akt-mT2 adenoviral infections, adenovirus was produced using the AdEasy protocol [257], prior to transducing the LSL-KRas^G12D/⁺, LSL-Trp53^R712H/⁺, Pdx1-Cre (KPC) pancreatic ductal adenocarcinoma (PDAC) cell line [258] in the presence of 8 μg/ml Polybrene. All microscopy was performed on a Leica DMI 6000 SP8 confocal microscope with a 25X water objective. Cells were given 48 h to express the biosensor before MP FLIM-FRET microscopy was performed at 840 nm with a Ti:Sapphire femtosecond

Author contributions

JRWC and SCW designed the study, performed experiments, and analyzed data. JRWC, SCW and PT wrote the manuscript. PT supervised the study.

Acknowledgements

The authors thank Dr. Andrew Burgess, Dr. Thomas R. Cox, Dr. Lucie Nedved, Dr. Max Nobis, Dr. David Herrmann and Kendelle J. Murphy for critical reading of the manuscript, Dr. Juliane Schwarz for the site-directed mutagenesis of BioSrc to BioSrcT, Dr. Marc Giry-Laterriere for subcloning of BioSrcT into pENTR2b and Claire Vennin for transfection and selection of KPC PDAC cells expressing BioSrcT.

This work was funded by an NHMRC project grant, an ARC Future grant, a Len Ainsworth Pancreatic

References (258)

K.E. Miller et al.
Bimolecular fluorescence complementation (BiFC) analysis: advances and recent applications for genome-wide interaction studies
J. Mol. Biol.
(2015)
S. Pennacchietti et al.
Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene
Cancer Cell
(2003)
P.P. Provenzano et al.
Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma
Cancer Cell
(2012)
C.W. Steele et al.
CXCR2 inhibition profoundly suppresses metastases and augments immunotherapy in pancreatic ductal adenocarcinoma
Cancer Cell
(2016)
S. Regot et al.
High-sensitivity measurements of multiple kinase activities in live single cells
Cell
(2014)
P.I. Merksamer et al.
Real-time redox measurements during endoplasmic reticulum stress reveal interlinked protein folding functions
Cell
(2008)
H.J. Snippert et al.
Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells
Cell
(2010)
A. Sakaue-Sawano et al.
Visualizing spatiotemporal dynamics of multicellular cell-cycle progression
Cell
(2008)
Z. Erami et al.
Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue
Cell Rep.
(2016)
M.A. Karreman et al.
Intravital correlative microscopy: imaging life at the nanoscale
Trends Cell Biol.
(2016)

M.T. Kunkel et al.

Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter

J. Biol. Chem.

(2005)

O. Gavet et al.

Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis

Dev. Cell

(2010)

D.C. Braun et al.

Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase Cdelta in the intact cell

J. Biol. Chem.

(2005)

M. Fehr et al.

In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors

J. Biol. Chem.

(2003)

D.S. Evanko et al.

Elimination of environmental sensitivity in a cameleon FRET-based calcium sensor via replacement of the acceptor with Venus

Cell Calcium

(2005)

K.M. Dean et al.

Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation

Biophys. J.

(2011)

E. Hirata et al.

Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin beta1/FAK signaling

Cancer Cell

(2015)

S. Sabiani et al.

Unraveling the activation mechanism of Taspase1 which controls the oncogenic AF4-MLL fusion protein

EBioMedicine

(2015)

J.P. Gardner et al.

Robust, but transient expression of adeno-associated virus-transduced genes during human T lymphopoiesis

Blood

(1997)

P. Horvath et al.

Screening out irrelevant cell-based models of disease

Nat. Rev. Drug Discov.

(2016)

J.R. Conway et al.

Developments in preclinical cancer imaging: innovating the discovery of therapeutics

Nat. Rev. Cancer

(2014)

D.R. Croucher et al.

Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers

Sci. Signal

(2016)

T.K. Kerppola

Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells

Nat. Protoc.

(2006)

G.S. Filonov et al.

A near-infrared BiFC reporter for in vivo imaging of protein-protein interactions

Chem. Biol.

(2013)

W.R. Wilson et al.

Targeting hypoxia in cancer therapy

Nat. Rev. Cancer

(2011)

B.G. Wouters et al.

Cells at intermediate oxygen levels can be more important than the “hypoxic fraction” in determining tumor response to fractionated radiotherapy

Radiat. Res.

(1997)

B.W. Miller et al.

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy

EMBO Mol. Med.

(2015)

M.A. Jacobetz et al.

Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer

Gut

(2013)

K.P. Olive et al.

Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer

Science

(2009)

T.R. Cox et al.

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase

Nature

(2015)

R. Straussman et al.

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion

Nature

(2012)

D.W. McMillin et al.

The role of tumour-stromal interactions in modifying drug response: challenges and opportunities

Nat. Rev. Drug Discov.

(2013)

C.D. Madsen et al.

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

EMBO Rep.

(2015)

T. Kitamura et al.

Immune cell promotion of metastasis

Nat. Rev. Immunol.

(2015)

R. Weissleder et al.

Imaging in the era of molecular oncology

Nature

(2008)

D.C. Swinney et al.

How were new medicines discovered?

Nat. Rev. Drug Discov.

(2011)

S.M. Paul et al.

How to improve R&D productivity: the pharmaceutical industry’s grand challenge

Nat. Rev. Drug Discov.

(2010)

A. Kamb

What’s wrong with our cancer models?

Nat. Rev. Drug Discov.

(2005)

B.A. Rabinovich et al.

Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer

Proc. Natl. Acad. Sci. U.S.A.

(2008)

C.P. Bracken et al.

Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion

EMBO J.

(2014)

A. Masedunskas et al.

Intravital microscopy: a practical guide on imaging intracellular structures in live animals

Bioarchitecture

(2012)

M. Canel 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.

(2010)

D. Kedrin et al.

Intravital imaging of metastatic behavior through a mammary imaging window

Nat. Methods

(2008)

L. Ritsma et al.

Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging

Nature

(2014)

J. Livet et al.

Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system

Nature

(2007)

A. Sakaue-Sawano et al.

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

Development

(2013)

Y.A. Yoo et al.

Bmi1 marks distinct castration-resistant luminal progenitor cells competent for prostate regeneration and tumour initiation

Nat. Commun.

(2016)

D.R. Chittajallu et al.

In vivo cell-cycle profiling in xenograft tumors by quantitative intravital microscopy

Nat. Methods

(2015)

A. Serrels et al.

Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development

Cancer Res.

(2009)

S.I. Ellenbroek et al.

Imaging hallmarks of cancer in living mice

Nat. Rev. Cancer

(2014)

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View full text

Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors

Highlights

Abstract

Introduction

Section snippets

Using fluorescent reporters to monitor therapeutic response

Considerations for IVM of live tumor xenografts

IVM equipment and analysis

Microenvironmental context for intravital imaging

Conclusions

Adenoviral FLIM-FRET imaging

Author contributions

Acknowledgements

J. Mol. Biol.

Cancer Cell

Cancer Cell

Cancer Cell

Cell

Cell

Cell

Cell

Cell Rep.

Trends Cell Biol.

J. Biol. Chem.

Dev. Cell

J. Biol. Chem.

J. Biol. Chem.

Cell Calcium

Biophys. J.

Cancer Cell

EBioMedicine

Blood

Screening out irrelevant cell-based models of disease

Nat. Rev. Drug Discov.

Developments in preclinical cancer imaging: innovating the discovery of therapeutics

Nat. Rev. Cancer

Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers

Sci. Signal

Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells

Nat. Protoc.

A near-infrared BiFC reporter for in vivo imaging of protein-protein interactions

Chem. Biol.

Targeting hypoxia in cancer therapy

Nat. Rev. Cancer

Cells at intermediate oxygen levels can be more important than the “hypoxic fraction” in determining tumor response to fractionated radiotherapy

Radiat. Res.

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy

EMBO Mol. Med.

Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer

Gut

Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer

Science

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase

Nature

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion

Nature

The role of tumour-stromal interactions in modifying drug response: challenges and opportunities

Nat. Rev. Drug Discov.

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

EMBO Rep.

Immune cell promotion of metastasis

Nat. Rev. Immunol.

Imaging in the era of molecular oncology

Nature

How were new medicines discovered?

Nat. Rev. Drug Discov.

How to improve R&D productivity: the pharmaceutical industry’s grand challenge

Nat. Rev. Drug Discov.

What’s wrong with our cancer models?

Nat. Rev. Drug Discov.

Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer

Proc. Natl. Acad. Sci. U.S.A.

Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion

EMBO J.

Intravital microscopy: a practical guide on imaging intracellular structures in live animals

Bioarchitecture

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.

Intravital imaging of metastatic behavior through a mammary imaging window

Nat. Methods