Bio-imaging of colorectal cancer models using near infrared labeled epidermal growth factor.

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues

Spectral intensity maps of BOI images of specific EGF-NIR binding in human CRC tissues.

<p>Images of five typical slices, specifically labeled with EGF-NIR, as described in legend for Fig. 7. The 800 nm Odyssey Infrared Imager acquired images were processed using applied spectral imaging software, Spectral View. Intensity scale-red-brown (5) high expression of EGF-NIR binding; green (4) intermediate expression; blue (3) very low expression.</p

The specificity and selectivity of EGF-NIR probe binding measured by IC-NIR imaging.

<p>HT-29 cells were incubated for 15 minutes at 37°C (A) and 4°C (B) with 7 nM EGF-NIR in the presence or absence of 100 nM EGF. Competition experiments with 500 nM of cetuximab, TGF-α or NRG1 were also conducted. In control experiments the cultures were incubated with 7 nM NIR-Dye to evaluate nonspecific labeling of the cells. The NIR intensity at 800 nm was estimated under identical conditions for all cultures and the mean ± SD (n = 9) is presented. Upper inserts: NIR scans; lower inserts: phase-contrast photomicrographs of the cultures,* p<0.05 vs. NIR-Dye; ** p<0.05 vs. EGF-NIR.</p

Saturation, kinetics and sensitivity of EGF-NIR binding using IC-NIR imaging of CRC cultures.

<p>(A) Left: a scheme of CRC polyp with high spots of transformed CRC cells (focal area) and heterogeneous area including both normal and transformed CRC cells; Middle: generation of a heterogeneous mixture at different ratios (%) between HT-29 and SW 620; 0 – no cells; +/++ -presence of different cell concentrations; Right: focal plating (in a ring) of HT-29 and A431 monolayer surrounded by SW620 monolayer; insert-the level of EGFR (170 kD) and non-mature EGFR (150 kD) in the cells; (B) The relationship between the NIR intensity of 15 min binding with 7 nM EGF-NIR (mean ± SD, n = 9) and the percentage of SW620 in the cell mixture with either HT-29 (closed circles) or HCT116 (open circles) * p<0.05 vs. 100% SW620; (C) The relationship between SBR (mean ± SD, n = 9) and EGF-NIR concentration; A431 (open circles); HT-29 (closed circles); binding was performed for 15 minutes. Insert: NIR scans; * p<0.05 vs. 0.01 nM; (D) The kinetics of 7 nM EGF-NIR binding (mean ± SD, n = 9) to focal cultures of A431 (open circles) or HT-29 (closed circles); Insert: NIR scans; * p<0.05 vs. 0 min.</p

Synthesis, purification, spectrum, electrophoresis properties and signaling of EGF-NIR.

<p>(A) Reaction scheme for the synthesis of EGF-NIR conjugate, the first amino acid asparagine at the amino terminal is indicated as Asn1. (B) Separation of synthesis reaction mixture on gel permeation chromatography and of EGF-NIR sample from gel permeation on (C) anion exchange chromatography; EGF-NIR-full line (800 nm); gradient of NaCl-broken line; unconjugated EGF-dotted line. (D) HPLC separation of EGF-NIR purified from anion exchanger chromatography. Full line represents absorbance at 226 nm and dotted line indicates the gradient. Insert-12% SDS-PAGE analysis of 10 µg of EGF-NIR scanned with Odyssey and unmodified EGF stained with coomassie blue. (E) NIR spectrum of EGF-NIR [excitation (gray line) and emission (black line)]; Insert-IRDye 800CW NHS ester; (F) EGF-NIR induced Erk phosphorylation.</p

BOI of EGF-NIR binding in human CRC tissues.

<p>36 Slices of CRC tissues and 19 slices of adjacent colon tissue (n = 10–15 ROI in each slice) were submitted for <i>ex vivo</i> binding assay for 45 min at 37°C with 70 nM EGF-NIR in the presence (non specific) or absence (total binding) of 1 µM unlabeled EGF. The NIR intensity was estimated at identical conditions for all slices (n = 12). Significance: * p<0.01 compared to respective group in adjacent colon EGFR-, ** p<0.05 compared to respective group in CRC tissue EGFR-; Insert: typical western blotting for EGFR of the slices investigated.</p

siRNA-induced knock down of EGFR evaluated by IC-NIR imaging.

<p>HT-29 cells were transfected for 2 days with 5 nM anti-EGFR Silencer select siRNA or scrambled RNA or left untreated (control). Evaluation of EGFR expression was performed by In Cell NIR imaging using 7 nM EGF-NIR (black bars) and western blotting (gray bars). The values are mean ± SD (n = 3). * p<0.05 vs. scrambled or control.</p

Whole body <i>in vivo</i> and isolated tissue <i>ex vivo</i> EGF-NIR BOI of mice with HT-29 orthotopic tumors.

<p>(A) Photograph of an orthotopic tumor and EGFR protein expression in the tumors; (B) Time course of EGF-NIR accumulation in tissues of tumor-bearing mice. The mice were injected i.v. with 1 nmol of EGF-NIR in untreated mice (upper row, n = 6) or mice pre-injected with 1 µg/ml cetuximab (lower row, n = 4); high resolution BOI of a mouse injected with EGF-NIR and circles indicate ROI measurements (C) Time course of tissue accumulation of EGF-NIR at 48 hours from mice presented in B; Signal intensity at 800 nm were normalized to background fluorescence using an arbitrary tumor circle (10–20 ROIs/mouse) compared to an identical area on the flank (adjacent muscle); * p<0.05 compared to EGF-NIR 4 hours; ** p<0.05 compared to mice injected with EGF-NIR; (D) EGF-NIR signal/background ratio in isolated tissue from the tumor-bearing mice 48 hours after injection. * p<0.05 compared to muscle, ** p<0.05 compared to liver; Insert: Upper-photographs of tissues in the dish; Middle-NIR images; Lower-spectral intensity maps; Intensity scale-red-brown (5) high expression; blue (3) very low expression.</p

IC-NIR BOI of EGFR levels in CRC clones, in relation to the level of expression of CEA.

<p>Different CRC cells were focally plated on a background of IEC6 enterocyte monolayer. The cultures were incubated for 15 min at 37°C with 7 nM EGF-NIR in the presence (nonspecific binding – white bars) or absence (total binding – grey bars) of 100 nM unmodified EGF. The signal (CRC cell line)/ background (IEC6) ratio was estimated at identical conditions for all cultures and is presented as the mean ± SD (n = 9). Significance: * p<0.05 compared to IEC6 values; ** p<0.05 compared to total binding of the respective group p< 0.05 compared to A431 and & p<0.05 compared to COLO 205; Lower inserts: NIR scans; Upper inserts: left-EGFR protein expression by western blotting; arrow indicate the position of mature EGFR 170 kD protein and non-glycosylated 150 kD protein; right-mRNA expression of CEA and β-actin in cell cultures.</p