Developing a fluorescent, tumour-specific, molecular imaging platform for laparoscopic colorectal cancer surgery.

Background: Fluorescent laparoscopic imaging of primary colorectal tumours and lymph node metastases would improve localisation of early tumours and allow intra-operative staging, potentially facilitating intra-operative stratification of the extent of surgical resections thereby improving patient outcomes. I aimed to develop and test a fluorescent molecular probe capable of allowing localisation of tumours in vivo. Methods: Immunohistochemistry was used to identify the most appropriate colorectal cancer target from a panel of seven biomarkers in prospectively collected, matched normal and tumour tissue from 280 colorectal cancer patients. The availability of targets for antibody binding was assessed in live cells, fresh frozen tumour samples and ex vivo cancer specimens. Indocyanine green was conjugated to anti-CEA and control IgG antibodies, and CEA-specific fluorescence was quantified in live cancer cells. Dye-doped silica nanoparticles loaded with NIR664 dye were synthesised using a water-in-oil microemulsion technique. Anti-CEA IgGs or control IgGs were conjugated to nanoparticles using a variety of different chemical conjugation strategies and binding to cells was quantified in vitro. A murine xenograft model and live IVIS imaging were used to assess PAMAM-linked nanoparticles in vivo. Results: Of the biomarkers tested, CEA showed the greatest differential expression between normal and tumour samples (p<0.001) and the best sensitivity (93.7%) and specificity (96.1%) for colorectal cancer detection. ICG-anti-CEA exhibited CEA-specific fluorescence in all three cell lines tested (p<0.01) with fluorescence peaking at 24-36 hours. CEA-targeted, PAMAM dendrimer-conjugated nanoparticles allowed strong tumour-specific targeting, demonstrating up to 12.3-fold greater fluorescence than control nanoparticles when incubated with colorectal cancer cell lines (p<0.002). In LS174T xenografts, CEA-targeted nanoparticles demonstrated clear tumour-specific fluorescence from 6 to 72h after injection, as compared to only background fluorescence for control IgG-targeted nanoparticles at all time points (p<0.0001). Conclusions: CEA has the greatest potential to allow highly specific tumour imaging. ICG-conjugated anti-CEA antibodies allow CEA-specific imaging of colorectal cancer cells in vitro. Dye-doped silica nanoparticles conjugated to anti-CEA antibodies via PAMAM dendrimers have potential to allow intra-operative fluorescent visualisation of tumour cells. These findings are the first to demonstrate live tumour-specific fluorescent colorectal cancer imaging using an antibody-targeted nanoparticle in vivo.