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Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor

Weitsman, Gregory; Mitchell, Nicholas J.; Evans, Rachel; Cheung, Anthony; Kalber, Tammy L.; Bofinger, Robin; Fruhwirth, Gilbert O.; Keppler, Melanie; Wright, Zoe V. F.; Barber, Paul R.; Gordon, Peter; Koning, Tamra de; Wulaningsih, Wahyu; Sander, Kerstin; Vojnovic, Borivoj; Hailes, Helen C.; Tabor, Alethea B.; Ng, Tony

Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor Thumbnail


Authors

Gregory Weitsman

Nicholas J. Mitchell

Rachel Evans

Anthony Cheung

Tammy L. Kalber

Robin Bofinger

Gilbert O. Fruhwirth

Melanie Keppler

Zoe V. F. Wright

Paul R. Barber

Peter Gordon

Tamra de Koning

Wahyu Wulaningsih

Kerstin Sander

Borivoj Vojnovic

Helen C. Hailes

Alethea B. Tabor

Tony Ng



Abstract

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.

Citation

Weitsman, G., Mitchell, N. J., Evans, R., Cheung, A., Kalber, T. L., Bofinger, R., …Ng, T. (in press). Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor. Oncogene, https://doi.org/10.1038/onc.2016.522

Journal Article Type Article
Acceptance Date Dec 21, 2016
Online Publication Date Feb 6, 2017
Deposit Date Mar 1, 2017
Publicly Available Date Mar 1, 2017
Journal Oncogene
Print ISSN 0950-9232
Electronic ISSN 0950-9232
Publisher Nature Publishing Group
Peer Reviewed Peer Reviewed
DOI https://doi.org/10.1038/onc.2016.522
Public URL https://nottingham-repository.worktribe.com/output/847566
Publisher URL http://www.nature.com/onc/journal/vaop/ncurrent/full/onc2016522a.html

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