Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor.
Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo.
Richard Dimbleby Department of Cancer Research, Randall Division &Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.
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G
Weitsman
Department of Chemistry, University College London, London, UK.
N J
NJ
Mitchell
Richard Dimbleby Department of Cancer Research, Randall Division &Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.
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R
Evans
Richard Dimbleby Department of Cancer Research, Randall Division &Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.|Breast Cancer Now Research Unit, King's College London, London, UK.
A
A
Cheung
UCL Centre for Advanced Biomedical Imaging, Division of Medicine, University College London, London, UK.
T L
TL
Kalber
Department of Chemistry, University College London, London, UK.
R
R
Bofinger
Richard Dimbleby Department of Cancer Research, Randall Division &Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.
G O
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Fruhwirth
Richard Dimbleby Department of Cancer Research, Randall Division &Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.
M
M
Keppler
Department of Chemistry, University College London, London, UK.
Z V F
ZVF
Wright
Gray Laboratories, Department of Oncology, Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Oxford, UK.
P R
PR
Barber
Breast Cancer Now Research Unit, King's College London, London, UK.
P
P
Gordon
Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.
T
T
de Koning
Cancer Epidemiology Group, Division of Cancer Studies, King's College London, London, UK.
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W
Wulaningsih
Institute of Nuclear Medicine, University College London, London, UK.
K
K
Sander
Gray Laboratories, Department of Oncology, Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Oxford, UK.
B
B
Vojnovic
Richard Dimbleby Department of Cancer Research, Randall Division &Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.
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0000-0001-5227-575X
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Ameer-Beg
UCL Centre for Advanced Biomedical Imaging, Division of Medicine, University College London, London, UK.
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M
Lythgoe
Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.
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JN
Arnold
Institute of Nuclear Medicine, University College London, London, UK.
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E
Årstad
King's College London Dental Institute, Tissue Engineering and Biophotonics, Guy's Hospital Campus, London, UK.
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F
Festy
Department of Chemistry, University College London, London, UK.
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HC
Hailes
Department of Chemistry, University College London, London, UK.
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Tabor
Richard Dimbleby Department of Cancer Research, Randall Division &Division of Cancer Studies, Kings College London, Guy's Medical School Campus, London, UK.|Breast Cancer Now Research Unit, King's College London, London, UK.|UCL Cancer Institute, Paul O'Gorman Building, University College London, London, UK.
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D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005453:Fluorescence; D006801:Humans; D008081:Liposomes; D008325:Mammary Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D059016:Tumor Microenvironment
D008081:Liposomes; D009363:Neoplasm Proteins; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C512478:EGFR protein, human; C541485:EGFR protein, mouse; D066246:ErbB Receptors