Stereomicroscopic fluorescence imaging of head and neck cancer xenografts targeting CD147

Abstract

Purpose: To demonstrate that systemically administered fluorescently labeled anti-CD147 antibody can detect head and neck squamous cell carcinoma xenografts in vivo.

Experimental design: In vivo immunodeficient murine model.

Results: Peak tumor fluorescence was visualized by near infrared stereomicroscopy in SCC-1 tumors at 24 hours after systemic injection of anti-CD147:Cy5.5 bioconjugate. SCC-1 xenografts demonstrated significantly higher fluorescent intensity after administration of CD147:Cy5.5 (48 au, p < 0.0001) compared to IgG1k:Cy5.5 isotype control antibody (9 au). FaDu tumors overexpressing CD147 (FaDu/E) demonstrated higher fluorescence (53 au) compared to control vector transfected cells (FaDu, 33 au, p < 0.0001) which was higher than CD147 knockdown cells (FaDu/siE, 5 au, p < 0.0001).

Methods: To determine if fluorescently labeled anti-CD147 antibody was specific for tumors in vivo, anti-CD147 and non-specific IgG1k antibody were labeled with a near infrared fluorophore (Cy5.5) and administered systemically to immunodeficient mice bearing SCC-1 xenografts. Imaging was performed over a 72 hour period using brightfield and fluorescent (685-735 nm) stereomicroscopy. To determine if fluorescence varied with receptor expression, SCID mice were xenografted with cell lines expressing variable amounts of CD147: FaDu (control vector transfected), FaDu/siE (siRNA CD147 knockdown) or FaDu/E (CD147 overexpressing) cells.

Conclusions: This data suggests fluorescently labeled anti-CD147 may have clinical utility in detection of HNSCC.

Figure 1

Digital and stereomicroscopic images of SCC-1 tumor xenografts (A–F) (n = 13) and engrafted human skin (G–I) (n = 4). SCID mice bearing xenografts underwent systemic tail vein injection of anti-CD147:Cy5.5 (100 µg) (n = 6) or nonspecific human IgG1:Cy5.5 antibody control (100 µg) (n = 7). Images were obtained at 24 and 48 hours. At 24 hours, tumor fluorescence with anti-CD147:Cy5.5 was 48 ± 9 arbitrary units (au), tumor fluorescence with IgG1k:Cy5.5 was 9 ± 4 au (p < 0.0001), and 22 ± 2 au (p < 0.0002) for human skin graft. Representative quantitative analysis of tumor illumination shown in graph form. (J) digital photograph of the Leica fluorescent stereomicroscope (Leica MZFL3 stereo research microscope, Leica Microsystems, Bannockburn, IL) fitted with Cy5.5 and GFP-2 filters and an ORCA ER charge-coupled device camera (Hamamatsu, Bridgewater, NJ) which was used for all imaging. All exposure times were 0.4 seconds. Bar = 2 mm.

Figure 2

Confocal and histologic images of SCC-1 tumor samples in the absence of anti-CD147:Cy5.5 (A and C), and after injection (B and D). Specimens were paraffin embedded and confocal images taken at 40× oil immersion lens after excitation with 633 nm light and emission captured at a range of 647–800 nm. Pseudo red coloring added after image capture. Bar = 25 µm.

Figure 3

Stereomicroscopic fluorescence of SCC-1 xenografts transfected to express green fluorescent protein (GFP). Tumors (n = 4) were imaged for Cy5.5 (A–D) and GFP (E–H) fluorescence. Overlays were then formed to demonstrate overlap of Cy5.5 and GFP fluorescence (I–L). (M) graphic illustration of the effects of time on Cy5.5 and GFP fluorescence. (N) using measurements of fluorescent areas, tumor areas measured using Cy5.5 were around 115% of areas measured using GFP through the first 72 hours post injection. Images were obtained at 24, 48, 72 and 144 hours after systemic injection of anti-CD147:Cy5.5 (100 µg). Exposure times were 0.2 sec for Cy5.5 and 0.1 sec for GFP. Bar = 2 mm.

Figure 4

(A–C) Stereomicroscopic fluorescence for Cy5.5 fluorescence. Images were obtained 24 hours after administering 200 µg anti-CD147 antibody at 0.5 sec exposure length. FaDu tumors overexpressing CD147 (FaDu/E) demonstrated higher fluorescence (53 ± 3 au) compared to control vector transfected cells (FaDu, 33 ± 3 au, p < 0.0001) which was higher than CD147 knockdown cells (FaDu/siE, 5 ± 3 au, p < 0.0001). (D–F) Immunohistochemistry images after staining tumor specimens for CD147. (G) Western blot analysis confirms overexpression of the EMMPRIN-green fluorescence protein (GFP) fusion protein. EMMPRIN expression was reduced in the FaDu/siE line. Control vector transfected cells (FaDu), expressed intermediate basal levels of EMMPRIN. Equal protein loading was confirmed by actin loading. (H) graphic representation of tumor fluorescence from 1, 6, 48, 72 and 144 hours post-injection of 200 µg of CD147:Cy5.5. Bar = 2 mm, Magnification = 40×.