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

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.

Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer

“…This association was also found to persist in the presence of an EGFR inhibitor. The same effect of macrophage infiltration on EGFR activation was also seen in colorectal cancer xenografts, but not in non-small-cell lung cancer xenografts expressing a constitutively activated EGFR mutant that could not be affected by macrophages (26).…”

“…Notably, because FLIM is less sensitive to the loss of donor emission intensity caused by scattering in tissues, FLIM-FRET can be effectively applied in vivo. For example, Weitsman et al (26) generated a FLIM-FRET-compatible version of the EGFR activity biosensor phosphorylation indicator of CrkII chimeric unit (Picchu) (27) by replacing YFP and CFP with mRFP1 and EGFP, respectively, and used the resulting Picchu-FLIM sensor to investigate EGFR activity in tumor xenografts. Another benefit of FLIM-FRET is that it does not require fluorescence emission by the acceptor, which can potentially alleviate some challenges of ratiometric imaging, such as spectral bleed-through.…”

“…Importantly, the failure of cells to respond to drugs could be linked to differences in the external microenvironment and not to EGFR expression. Therefore, Weitsman et al (26) utilized their FLIM-FRET-based EGFR biosensor Picchu-FLIM, which was delivered into a murine model of basallike breast cancer in vivo via liposomes, to examine the intratumoral heterogeneity of EGFR activity. This work revealed substantial heterogeneity in both EGFR activity and EGFR inhibitor efficacy in a murine breast cancer model.…”

Development of lipopolyplexes for gene delivery: A comparison of the effects of differing modes of targeting peptide display on the structure and transfection activities of lipopolyplexes

“…27 We have recently used this approach to formulate lipopolyplexes that selectively transfected tumor cells with pDNA coding for a FRET biosensor and used this to monitor EGFR inhibition by tyrosine kinase inhibitors in vivo using quantitative FRET-FLIM imaging. 28 In this work, the bifunctional peptide incorporated peptide sequences targeting EGFR, conferring tumor selectivity and active targeting on these lipopolyplexes.…”

“…We have also shown that liposomes formulated including these n‐EG lipids form nanoparticles that are shielded with a shallow, homogeneous n‐EG layer, and that these have much better cellular uptake than liposomes formulated with 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐ N ‐[carboxy (polyethyleneglycol) 2000 ] (DSPE‐PEG2000) . We have recently used this approach to formulate lipopolyplexes that selectively transfected tumor cells with pDNA coding for a FRET biosensor and used this to monitor EGFR inhibition by tyrosine kinase inhibitors in vivo using quantitative FRET‐FLIM imaging . In this work, the bifunctional peptide incorporated peptide sequences targeting EGFR, conferring tumor selectivity and active targeting on these lipopolyplexes.…”