Affinity of FED6 and uptake of [¹⁸F]FED6 in NSCLC cell lines.

A. IC50 values of FED6 obtained from measuring changes in p-EGFR Y1068 in NSCLC cell lines following incubation with FED6 for 3 h. Data are mean IC50 ± SE obtained from three separate experiments. B. Protein normalised uptake of [18F]FED6 in NSCLC cell lines following 1 h incubation with 0.22MBq [18F]FED6. C. Protein expression of total EGFR, and ABC transporters ABCG2 and ABCB1 in NSCLC cells. MCF7MX and 3T3-MDR1 were used as positive controls for ABCG2 and ABCB1 expression, respectively.</p

[¹⁸F]FED6 uptake in ABC transporter expressing cells.

A. Protein normalised uptake of [18F]FED6 in 3T3 and 3T3-MDR1 cells, following 1 h incubation with 0.22MBq of [18F]FED6, with or without pre-treatment with zosuquidar at 0.3 μM for 1 h. B. Protein normalised uptake of [18F]FED6 in MCF7 and MCF7MX cells, following 1 h incubation with 0.22MBq of [18F]FED6, with or without pre-treatment with 10 μM fumitremorgin C (FTC) for 1 h. Data are mean ± SE of triplicates, repeated twice. Statistical differences between data obtained following an unpaired t-test are indicated on graph; (** = P < 0.01).</p

Qualitative comparison of <i>in vitro</i> binding of NT-MB and T140-MB to CXCR4-expressing cells.

(A) Brightfield images of T140-MB and NT-MB binding to MDA-MB-231 cells transduced with a non-coding (shSC) or a CXCR4-targeted shRNA (shCXCR4) in the presence of vehicle, DOX (0.5 μg/mL for 24h), or blocking with free T140 peptide (1 mg/mL for 5 min). Images were obtained under 400x magnification and scale bar represents 100 μm. (DOCX)</p

Development of a CXCR4-targeted microbubble.

(A) Scheme of synthesis of DSPE-PEG2000-T140 lipo-PEG-peptide through covalent coupling of the TN14003 peptide with the DSPE-PEG2000-NH2 lipid. (B) Lipid composition of NT-MB and T140-MB expressed in mole fraction (%). Microbubble concentration (C) and size (D) were determined using dedicated software for analysis of optically acquired FOV as exemplified in (E). Data represents mean ± SEM, n = 4. Images were obtained under 400x magnification and scale bar represents 100 μm. (F) Typical appearance of NT-MB and T140-MB vials after shaking. (F) Cell membrane integrity analysis by measuring LDH released into the supernatant by HepG2 cells upon treatment with indicated doses of microbubble T140-lipid mix and non-targeted lipid mix for 4 hours. The LDH provided by the manufacturer was used as the positive control. The data are presented as mean ± SD, n = 6; **P < 0.01.</p

T140-MB binds sensitively and specifically to CXCR4-expressing cells.

(A) Brightfield, fluorescent and merged images of T140-MB modified with 2 μg/mL of DiLC18(3) and (B), of T140-MB binding to MDA-MB-231 shCXCR4 cells in the presence of vehicle, DOX (0.5 μg/mL for 24h), or blocking with free T140 peptide (1 mg/mL for 5 min). (C) Quantification of microbubble fluorescence normalized to cell density. Data represents mean ± SEM, n = 6. (D) Confirmation of CXCR4 knockdown in the presence of DOX determined by western blot. β-actin was used as loading control. Images were obtained under 200x magnification and scale bar represents 100 μm.</p

S1 Raw images -

(PDF)</p

The effect of cyanoquinoline inhibitors on growth of ABC transporter-proficient or ABC transporter-deficient cell lines.

<p>The effect of cyanoquinoline inhibitors on growth of ABC transporter-proficient or ABC transporter-deficient cell lines.</p

Growth and necrosis in tumours.

(A) Ultrasound images show a dark region without perfusion, indicating some degree of necrosis, which was confirmed by haematoxylin and eosin staining shown in (B). (C) Growth curve of U2932 and SuDHL8 tumours determined by regular measurements of volume by callipers. Arrows indicate tumour edges and scale bar represents 1 mm in (A) and 100 μm in (B). (DOCX)</p

Constituents of T140-MB formulation.

(DOCX)</p

Comparative affinities FED20, gefitinib and FED6 in TKI sensitive and resistant NSCLC cell lines and [¹⁸F]FED20 uptake in the cell lines.

<p>Typical of p-EGFR Y1068 and EGFR protein expression following 3 h incubation with increasing concentrations of A. FED20, B. gefitinib, or C. FED6 in PC9 and PC9ER cells; summary IC50 values for all cell lines determined from densitometry analysis of p-EGFR Y1068 in the western blots are shown. D. Protein normalised uptake of [¹⁸F]FED20 in TKI sensitive and resistant cells following 1 h incubation with 0.55 MBq of [¹⁸F]FED20 (low specific activity; 7.3 GBq/μmol). E. Protein normalised uptake of [¹⁸F]FED20 in TKI sensitive and resistant cells following 1 h incubation with 0.74 MBq of [¹⁸F]FED20 (higher specific activity; 52 GBq/μmol). (* = p < 0.05, ** = P < 0.01).</p