Monitoring of Gefitinib Sensitivity with Radioiodinated PHY Based on EGFR Expression

Abstract

Epidermal growth factor receptor (EGFR) is attractive target for tumor diagnosis and therapy, as it is specifically and abundantly expressed in tumor cells. EGFR-tyrosine kinase (TK) inhibitors such as gefitinib and erlotinib are widely used in the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated whether radioiodinated 4-(3-iodo-phenoxy)-6,7-diethoxy-quinazoline (PHY), which is a candidate EGFR-TK imaging agent for single photon emission computed tomography (SPECT) is able to predict gefitinib sensitivity. We used four NSCLC cell lines-A549 (wild-type EGFR), H1650 (mutant EGFR; del E746_A750), H1975 (mutant EGFR; L858R, T790M) and H3255 (mutant EGFR; L858R)-and one epidermoid carcinoma cell line, A431 (wild-type EGFR). Cell proliferation assay and Western blotting revealed that A431 and H3255 with high EGFR expression showed high sensitivity to gefitinib. On the other hand, A549, H1650 and H1975 showed much lower sensitivity to gefitinib. The blocking study revealed that gefitinib decreased tumor uptake in ¹²⁵I-PHY in A431-bearing mice. Moreover, in vivo tumor uptake of ¹²⁵I-PHY was correlated with the IC₅₀ of gefitinib for cell proliferation. In the present study, tumor uptake of ¹²⁵I-PHY was correlated with the gefitinib sensitivity and this uptake was based on expression levels of EGFR, but not on mutation status. Although the mutation status is the most important factor for predicting gefitinib sensitivity, the abundant expression of EGFR is essential for therapy with EGFR-TK inhibitors. Therefore, radioiodinated PHY is a potential imaging agent to predict gefitinib sensitivity based on EGFR expression levels though further modifications of the imaging agent is needed to accurately estimate the mutation status.

Epidermal growth factor receptor (EGFR) is overexpressed in a variety of tumors including non-small cell lung cancer (NSCLC), head and neck cancer, glioma and ovarian cancer.^1,2) Activation of EGFR tyrosine kinase (EGFR-TK) is involved in the proliferation, invasion, metastasis, angiogenesis and suppression of apoptosis.³⁾ Therefore, EGFR is an attractive target molecule for tumor diagnosis and therapy.

To date, numerous EGFR-TK inhibitors and anti-EGFR antibodies have been developed and used in clinical settings.^2,4) Gefitinib, an EGFR-TK inhibitor, has been approved as a molecular targeted agent for the treatment of advanced NSCLC. Gefitinib leads to rapid symptom improvement and tumor regression in patients with NSCLC. Tumor responses were, however, observed in only 10–19% of patients with NSCLC in the phase II trial.^5,6) In addition, interstitial lung disease is a serious and fatal adverse effect of gefitinib treatment.⁷⁾ Retrospective epidemiologic analyses have revealed that the therapeutic effects are more apparent in Japanese, women and non-smokers.⁸⁾ From recent molecular biological studies,^9–11) mutations in the TK domain of EGFR gene are closely associated with gefitinib sensitivity. Therefore, development of diagnostic technology is necessary to predict or estimate therapeutic efficacy by EGFR-TK inhibitors.

Nuclear medicine techniques (positron emission tomography; PET and single photon emission computed tomography; SPECT) are able to estimate physiological and biological functions at the molecular or gene levels, as well as pharmacokinetics in vivo.^12,13) Radiolabeled inhibitors are believed to clarify pharmacokinetics, expression levels and functions of target molecules, and to estimate the therapeutic efficacy of the inhibitors. Although ¹⁸F-gefitinib was investigated to monitor EGFR expression and mutation status, tumor uptake was not correlated with EGFR expression levels and mutation status.¹⁴⁾ Development of various EGFR-TK imaging agents based exclusively on the anilinoquinazoline structure has been attempted for more than a decade, but further optimization of the properties of imaging agents with respect to high specificity to EGFR, selectivity to mutant types of kinase and high tumor uptake is required.^15–20) Thus, novel imaging agents to accurately detect EGFR-TK activity are needed in order to predict and monitor the therapeutic effects.

We have reported that radioiodinated 4-(3-iodo-phenoxy)-6,7-diethoxy-quinazoline (PHY, Fig. 1) is a candidate EGFR-TK imaging agent for SPECT.²¹⁾ In this report, we measured the uptake of ¹²⁵I-PHY in tumors with various expression levels of EGFR and types of mutation, and investigated whether gefitinib sensitivity can be estimated with radioiodinated PHY.

Fig. 1. Chemical Structures of 4-(3-Iodo-phenoxy)-6,7-diethoxy-quinazoline (PHY), PD153035 and Gefitinib

MATERIALS AND METHODS

Cell Culture

Four NSCLC cell lines-A549 (wild-type EGFR), H1650 (mutant EGFR; del E746_A750), H1975 (mutant EGFR; L858R, T790M) and H3255 (mutant EGFR; L858R)-and one epidermoid carcinoma cell line, A431 (wild-type EGFR), were used in this study (Table 1). A431 and A549 were obtained from DS Pharma Biomedical (Osaka, Japan). H1650 and H1975 were purchased from the American Tissue Culture Collection (Manassas, VA , U.S.A.). H3255 was kindly provided by Dr. Juri Gelovani (M.D. Anderson Cancer Center). A431 and A549 were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, U.S.A.) supplemented with 10% fetal bovine serum (FBS; Invitrogen Inc., Carlsbad, CA, U.S.A.). H1650 and H1975 were maintained in RPMI1640 (Sigma) supplemented with 10% FBS and 2 mM glutamine. H3255 was maintained in DMEM/F12 (Sigma) supplemented with 20% FBS. All cells were cultured in a 5% CO₂-humidified atmosphere at 37°C.

Table 1. Characterization and Sensitivity to Gefitinib in Tumor Cell Lines Used in This Study

Cell lines	Origin	EGFR status	IC50 (μM)
A431	Human epidermoid carcinoma	Wild-type	1.8
A549	Human NSCLC (Adenocarcinoma)	Wild-type	17.4
H1650	Human NSCLC (Adenocarcinoma)	del E746-A750	12.1
H1975	Human NSCLC (Adenocarcinoma)	L858R, T790M	13.3
H3255	Human NSCLC (Adenocarcinoma)	L858R	0.02

Growth Inhibition Assay

Growth inhibition by gefitinib was assessed using CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, U.S.A.). Cells were seeded in 96-well plates and incubated in culture media overnight at 37°C. The numbers of cells per well used in these experiments were as follows: 4000 for A431; 1000 for A549; 3000 for H1650; 2500 for H1975; and 5000 for H3255. After incubation with gefitinib, PD153035 and PHY at concentrations ranging from 1 nM to 33.3 µM for 72 h, 20 µL of CellTiter 96® AQ_ueous One Solution was added to each well and plates were incubated for a further 2 h at 37°C. Absorbance was measured at 490 nm using a micro-plate reader. Each experiment was set up in four replicate wells for each drug concentration and was repeated at least 4 times. IC₅₀ values were determined using a nonlinear regression model with a sigmoidal dose response (GraphPad Prism version 4.00 for Widows; GraphPad Software, San Diego, CA, U.S.A.).

Western Blotting

Cells were lysed in radio immunoprecipitation assay (RIPA) buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid sodium salt, 0.1% sodium dodecyl sulfate (SDS)] containing protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor cocktail (Nacalai Tesque). Protein concentration was determined using BCA Protein Assay Reagent kit (Thermo-Fisher Scientific, Rockford, IL, U.S.A.). Lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-FL, Millipore, Billerica, MA, U.S.A.). Membranes were blocked for 1 h in LI-COR blocking buffer and were incubated with primary and secondary antibodies. Rabbit polyclonal anti-EGFR (SC-03) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), anti-phospho-EGFR (Tyr1068) (Cell Signaling Technology, Danvers, MA, U.S.A.) and mouse monoclonal anti-β actin (Sigma) were used as primary antibodies. Alexa Fluor 680 goat anti-rabbit immunoglobulin G (IgG) (Molecular Probes, Eugene, OR, U.S.A.) and IRDye 800 anti-mouse IgG (Rockland Immunochemicals, Gilbertsville, PA, U.S.A.) were used as secondary antibodies. Membranes were scanned and bands were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, U.S.A.).

Tumor Xenograft Model

Animal studies were performed in compliance with the guidelines for the care and use of laboratory animals of Kanazawa University. Biodistribution studies were conducted in nude mice bearing tumors. Female BALB/c nu/nu mice aged 5–6 weeks (Japan SLC, Inc. Hamamatsu, Japan) were xenografted s.c. in the right and left dorsum with 2–5×10⁶ cells. Tumor size was below 1 cm in diameter.

Radiosynthesis of ¹²⁵I-PHY

¹²⁵I-NaI (MP Biomedicals, Santa Ana, CA, U.S.A.) and 10 µL of 0.1 N HCl were added to 20 µL of tributylstannyl precursor (1 mg/mL in EtOH), followed by addition of 10 µL of 30% hydrogen peroxide. After mixing for 5 min, the reaction was quenched by adding 10 µL of 10% sodium metabisulfate. Purification of ¹²⁵I-PHY was performed by HPLC using a Cosmosil 5C₁₈-AR II column (10×250 mm; Nacalai Tesque) eluted with 85% MeOH and 15% water at a flow rate of 2.0 mL/min.

Biodistribution of ¹²⁵I-PHY in Tumor-Bearing Mice

Tumor-bearing mice were injected via the tail vein with 74 kBq/100 µL (saline) of ¹²⁵I-PHY. Mice were sacrificed at 1, 4 and 24 h post-injection, and tissue samples were excised. To investigate EGFR-TK-specific uptake of ¹²⁵I-PHY, gefitinib (10 mg/kg) was co-injected with ¹²⁵I-PHY. Biodistribution was determined at 4 h post-injection. Tissue samples were weighed and radioactivity was measured with a γ-counter (ARC-360, Aloka, Tokyo, Japan). Uptake in organs was expressed as the percentage of injected dose per gram of tissue (%ID/g).

Statistical Analysis

Data analysis was performed using GraphPad Prism. Unpaired t-test was used for the blocking study. The correlation between IC₅₀ of gefitinib and tumor uptake of ¹²⁵I-PHY was tested using Spearman correlation test. The results were considered statistically significant at p<0.05.

RESULTS

Expression of EGFR and Phospho-EGFR and Growth Inhibition by Gefitinib

We used five tumor cell lines (2 wild-type EGFR and 3 mutant EGFR) and analyzed EGFR and phosphorylated EGFR (phospho-EGFR) expression levels by western blotting (Fig. 2). IC₅₀ values for growth inhibition by gefitinib were also measured by MTS assay (Table 1). There was high expression of EGFR and phospho-EGFR in A431 and H3255, whereas the IC₅₀ of H3255 (L858R) (0.02 µM) was much lower than that of A431 (1.8 µM). The highest IC₅₀ was observed in A549 (17.4 µM), although the expression of EGFR and phospho-EGFR was moderate. H1650 (del E746-A750) and H1975 (L858R+T790M) showed lower expression of EGFR and phospho-EGFR, and the IC₅₀ values for H1650 and H1975 were 12.1 µM and 13.3 µM, respectively.

Fig. 2. Expression of EGFR and Phospho-EGFR, as Detected by Western Blotting

Effect of L858R Mutation on Growth Inhibition by EGFR Inhibitors

In order to investigate whether EGFR mutations affect growth inhibition by PHY and gefitinib, A431 and H3255 were selected because they have abundant expression of EGFR and show different gefitinib sensitivity. The IC₅₀ of PD153035, which is an analogue of PHY, was also measured to investigate the structure–activity relationship. All compounds showed potent growth inhibition in H3255 (Table 2). Increased inhibitory effects due to this mutation were only observed in gefitinib treatment. The IC₅₀ in H3255 was more than 90-fold lower than that in A431, while the IC₅₀ values for PD153035 and PHY in H3255 were only 5.6- and 3.1-fold lower than those in A431.

Table 2. IC₅₀ of Gefitinib, PD153035 and PHY against Cell Growth in A431 (Wild EGFR) and H3255 (L858R)

	A431	H3255	A431/H3255
Gefitinib (µM)	1.80	0.02	90.0
PD153035 (µM)	1.63	0.29	5.6
PHY (µM)	6.50	2.10	3.1

Biodistribution of ¹²⁵I-PHY in A431-Bearing Mice

Table 3 shows the biodistribution of ¹²⁵I-PHY in A431-bearing mice. ¹²⁵I-PHY showed significant uptake in the intestine (30.6%ID/g at 1 h) and moderate uptake in the liver and stomach (3.3%ID/g and 4.6%ID/g at 1 h, respectively). There was low uptake of ¹²⁵I-PHY in other normal tissues. ¹²⁵I-PHY was rapidly cleared, whereas only the uptake in thyroid slowly increased up to 24 h post-injection (0.27%ID at 1 h to 0.82%ID at 24 h). Figure 3 indicates the effects of gefitinib on ¹²⁵I-PHY uptake at 4 h post-injection. Co-injection of gefitinib with ¹²⁵I-PHY resulted in a significant decrease in uptake in A431 (0.50%ID/g for control and 0.26%ID/g for gefitinib blocking).

Table 3. Biodistribution of ¹²⁵I-PHY in A431-Bearing Nude Mice (n=4)

	1 h	4 h	24 h
Blood	0.72±0.06	0.62±0.09	0.03±0.01
Brain	0.24±0.02	0.06±0.01	0.00±0.00
Liver	3.30±1.15	1.62±0.78	0.07±0.02
Heart	0.65±0.10	0.27±0.04	0.02±0.01
Lung	1.08±0.17	0.51±0.04	0.06±0.04
Stomach	4.60±1.82	2.90±1.64	0.13±0.08
Kidney	1.33±0.16	0.62±0.06	0.03±0.01
Pancreas	0.84±0.13	0.31±0.07	0.01±0.01
Spleen	0.52±0.10	0.22±0.04	0.02±0.02
Muscle	1.12±0.39	0.23±0.01	0.05±0.06
Thyroid (%ID)	0.27±0.08	0.68±0.13	0.82±0.13
Intestine	30.57±6.07	22.70±5.14	0.92±0.55
A431	1.56±0.25	0.47±0.12	0.03±0.01

Data are expressed as %ID/g (mean±S.D.).

Fig. 3. Biodistribution of ¹²⁵I-PHY at 4 h with and without Co-injection of Gefitinib (n=3–4)

Radioactivity in tissues is expressed as %ID/g (mean±S.D.). * p<0.05 vs. control.

Relationship between Tumor Uptake of ¹²⁵I-PHY and Gefitinib Sensitivity

¹²⁵I-PHY accumulated at various levels in tumors with different EGFR expression levels, as shown in Fig. 4 (1.56%ID/g for A431, 1.04%ID/g for A549, 1.31%ID/g for H1650, 0.94%ID/g for H1975 and 2.04%ID/g for H3255). Tumor uptake was reduced to less than half at 4 h post-injection. Tumors to blood (T/B) ratio and tumor to muscle (T/M) ratios were entirely good at 1 h post-injection (Fig. 5). A431, H1650 and H3255 showed good tumor to lung (T/L) ratios. As shown in Fig. 6, there is a significant correlation between ¹²⁵I-PHY uptake and gefitinib sensitivity (IC₅₀) (r=−0.900, p<0.05).

Fig. 4. In Vivo Uptake of ¹²⁵I-PHY in Tumor Bearing Mice (n=4)

Fig. 5. Tumor to Blood (T/B), Muscle (T/M) and Lung (T/L) Ratios of ¹²⁵I-PHY

Fig. 6. Correlation between in Vivo Tumor Uptake at 1 h and Gefitinib Sensitivity (r=−0.900, p<0.05)

DISCUSSION

We investigated the correlation between in vivo tumor uptake of ¹²⁵I-PHY and gefitinib sensitivity. In this study, we used one epidermoid carcinoma cell line and four NSCLC cell lines with different levels of EGFR expression and mutation status. These cell lines showed a range of gefitinib sensitivities. The present study indicated that the radioiodinated PHY is able to estimate gefitinib sensitivity based on EGFR expression levels.

Biodistribution studies indicated that ¹²⁵I-PHY is significantly taken up in the intestine, followed by the stomach and liver. This biodistribution was similar to that of other EGFR-TK imaging agents.^14,19) One preferable characteristic is that ¹²⁵I-PHY uptake in blood, lung and brain is low, resulting in relatively high T/B and T/L ratios. This suggests that radioiodinated PHY is useful for imaging lung and brain tumors.

The in vivo tumor uptake of ¹²⁵I-PHY is EGFR-TK specific and is negatively correlated with the IC₅₀ values of gefitinib in five cell lines. These results are in accordance with the previous results¹⁸⁾ that indicate correlations between ¹¹C-PD153035 uptake and EGFR expression levels, as measured by flow cytometry. PHY is structurally similar to PD153035 and the inhibitory potency of PHY against EGFR-TK phosphorylation is the same as that of PD153035.

Some tumor uptake is caused by nonspecific binding of ¹²⁵I-PHY, most likely to the cell membrane. In A431 and A549 harboring wild-type EGFR, gefitinib sensitivity of A431 was 10-fold higher than that of A549 and western blotting also showed abundant expression of EGFR in A431 when compared with A549, while ¹²⁵I-PHY uptake in A431 was only 1.5-fold higher than that in A549. An excess of gefitinib reduced ¹²⁵I-PHY uptake in A431 up to 52%. Therefore, reduction in nonspecific binding would result in estimating EGFR expression in more detail.

Although radiolabeled gefitinib should be suitable for estimating gefitinib sensitivity and assessing mutation status, ¹⁸F-gefitinib did not reflect EGFR expression levels.^{14) 125}I-PHY and ¹¹C-PD153035 were washed out of tumors, whereas ¹⁸F-gefitinib was retained in tumors throughout the experiment. These observations suggest that nonspecific binding of ¹⁸F-gefitinib to the cell membrane predominantly contributes to tumor uptake. In comparison with gefitinib, therefore, PHY and PD153035 may successfully penetrate into the cell membrane and then bind to EGFR-TK.

Estimation of EGFR expression would be a useful index for selecting patients to benefit from treatment with EGFR-TK inhibitors and to monitor inhibition status. Several groups^9–11) have shown that mutations in the TK domain of the EGFR gene are significantly correlated with clinical response to gefitinib therapy. Therefore, this mutation is a candidate to predict gefitinib sensitivity. Unfortunately, there are no significant differences in tumor uptake and clearance of ¹²⁵I-PHY between cells with wild-type EGFR and mutant EGFR. However, the overexpression is a prerequisite for treatment with EGFR-TK inhibitors.

Imaging agents are required to detect mutation status for accurate prediction of therapeutic efficacy with EGFR-TK inhibitors. The level of EGFR expression in tumors is, in itself, insufficient to account for their sensitivity to treatment.^22,23) PHY and PD153035 showed slight enhancement of growth inhibition in H3255, with the IC₅₀ values for PHY and PD153035 being 3.1- and 5.6-fold lower than those in A431, respectively. In contrast, gefitinib showed marked growth inhibition in H3255, in which the IC₅₀ of gefitinib is 90-fold lower than in A431. Crystal structure analysis^24,25) revealed that substitution of Leu858 with arginine maintains kinase activity in the active state and allows gefitinib to move deeper inside the binding pocket. Although ¹⁸F-gefitinib is unsuitable for imaging EGFR-TK activity, these findings suggest that introduction of propylmorpholino or other substituents to the 6 position of quinazoline is required for assessing mutation status.

CONCLUSION

Although EGFR expression levels are not sufficient to predict treatment efficacy with EGFR-TK inhibitors, EGFR overexpression in tumors is an essential factor for EGFR targeted therapy. Our study suggested that radioiodinated PHY is a potential imaging agent to detect EGFR expression levels as a first screening marker and we require further modifications of the compound to accurately estimate gefitinib sensitivity.

Acknowledgment

This work was supported by Grants-in-Aid for Scientific Research (C) No.10770466, No.19591437, (B) No.18390331 from JSPS and a Grant-in-Aid for High Technology Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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