Epidermal growth factor receptor-targeted molecular imaging of colorectal tumors: Detection and treatment evaluation of tumors in animal models

Abstract

To overcome the problem of overlooking colorectal tumors, a new and highly sensitive modality of colonoscopy is needed. Moreover, it is also important to establish a new modality to evaluate viable tumor volume in primary lesions of colorectal cancer (CRC) during chemotherapy. Therefore, we carried out molecular imaging of colorectal tumors targeting epidermal growth factor receptor (EGFR), which is highly expressed on tumor cells, for evaluating chemotherapeutic efficacy and for endoscopic detection of colorectal adenomas. We first attempted to image five CRC cell lines with various levels of EGFR expression using an Alexa Fluor-labeled anti-EGFR monoclonal antibody (AF-EGFR-Ab). A strong fluorescence signal was observed in the cells depending on the level of EGFR expression. When nude mice xenografted with LIM1215 CRC cells, which highly express EGFR, were i.v. injected with AF-EGFR-Ab, a strong fluorescence signal appeared in the tumor with a high signal to noise ratio, peaking at 48 hours after injection and then gradually decreasing, as shown using an IVIS Spectrum system. When the xenografted mice were treated with 5-fluorouracil, fluorescence intensity in the tumor decreased in proportion to the viable tumor cell volume. Moreover, when the colorectum of azoxymethane-treated rats was observed using a thin fluorescent endoscope with AF-EGFR-Ab, all 10 small colorectal adenomas (≤3 mm) were detected with a clear fluorescence signal. These preliminary results of animal experiments suggest that EGFR-targeted fluorescent molecular imaging may be useful for quantitatively evaluating cell viability in CRC during chemotherapy, and also for detecting small adenomas using a fluorescent endoscope.

Keywords: colorectal cancer; epidermal growth factor receptor; fluorescence endoscopy; molecular imaging; therapeutic efficacy.

Figure 1

Cellular imaging and fluorescence intensity in various colorectal cancer (CRC) cell lines. A, Five CRC cell lines (M7609, LIM1215, HT‐29, DLD‐1, COLO320DM) were incubated with Alexa Fluor 488‐labeled mouse antihuman epidermal growth factor receptor (EGFR) monoclonal antibody (AF488‐EGFR‐Ab), and then observed by confocal laser microscopy. Alexa Fluor 488‐labeled normal mouse IgG2a was used as a negative control. DAPI was used for nuclear staining. B, Fluorescence intensity and number of EGFR in each cell line were determined as described in Materials and Methods2. Correlation between fluorescence intensity and number of EGFR was assessed by Pearson's correlation test

Figure 2

Chronological changes of in vivo molecular imaging of LIM1215 and COLO320DM xenograft tumors in nude mice. A, Nude mice xenografted with LIM1215 or COLO320DM cells were injected with AF647‐epidermal growth factor receptor (EGFR)‐Ab into the tail vein, and tumors were observed using an IVIS Spectrum system (Perkin Elmer, Waltham, MA, USA). Alexa Fluor 647‐labeled normal mouse IgG2a was used as a negative control. B, Mean fluorescence intensity of the tumors from three mice (± SD) at each timepoint is shown

Figure 3

In vivo molecular imaging of LIM1215 xenograft tumor treated with fluorouracil (5‐FU). A, Mice were i.p. treated three times with 5‐FU or vehicle alone as described in Figure S1a, and then injected with AF647‐epidermal growth factor receptor (EGFR)‐Ab, after which fluorescence imaging was done using an IVIS Spectrum system (Perkin Elmer, Waltham, MA, USA). Representative images of tumors in mice treated with vehicle alone or 5‐FU 48 h after giving AF647‐EGFR‐Ab are shown. B, Mean fluorescence intensity (± SD) of the tumors observed in five mice at each timepoint is shown. *P < .01 by Student's t test

Figure 4

Relationship between epidermal growth factor receptor (EGFR) fluorescence intensity and tumor viability with or without fluorouracil (5‐FU) treatment. A, Tumor volumes of xenografts in each mouse (n = 8) before and after 5‐FU treatment were plotted. B, Fluorescence intensity of the xenograft tumor before and after 5‐FU treatment in each mouse was plotted. C‐F, Representative H&E and carcinoembryonic antigen (CEA) staining patterns in the tumor after vehicle alone or 5‐FU treatment. H&E staining after vehicle treatment (C), or 5‐FU treatment (D), immunohistochemical staining for CEA after vehicle treatment (E), or 5‐FU treatment (F) is shown. G, CEA‐positive rates in the tumor after vehicle treatment and 5‐FU treatment were plotted. *P < .01 by Student's t test. H, Correlation between the percentage decrease of the CEA‐positive rate and percentage decrease of fluorescence intensity in each tumor was assessed by Pearson's correlation test

Figure 5

Endoscopic and histological findings of colorectal polyps from azoxymethane (AOM)‐treated rats. A,B, Representative endoscopic image of a colorectal polyp in an AOM‐injected rat (rats #1, #3) observed using a thin endoscope. C,D, A 3‐mL aliquot of AF488‐epidermal growth factor receptor (EGFR)‐Ab (20 μg/mL) was given by enema with or without pretreatment with non‐labeled EGFR‐Ab (200 μg/mL), and colorectal polyps were detected by fluorescent thin endoscopy. E, The entire colorectum was removed from the rat. Yellow arrows show a small polyp. F, Histopathological findings of the resected tumor (H&E). G, EGFR immunohistochemical staining of the polyp. H, Treatment with normal rabbit IgG as a negative control