Introduction: Extent of resection contributes to glioblastoma multiforme (GBM) patient outcomes. Fluorescence-guided surgery increases the rate of gross total resection. CLR1501 (Novelos, Inc), a novel green fluorescent phospholipid ether analog, is selectively retained in cancer cells and was evaluated for GBM cellular imaging.
Methods: Five human-derived glioblastoma stem cell (GSC) enriched tumor lines and five GBM lines were used for in vitro studies, with comparison to normal human astrocytes (NHA). Cells were treated with CLR1501 (10 uM) and fluorescence quantified using flow cytometry. NOD-SCID mice with orthotopic GSC implants were used for evaluation of in vivo CLR1501 labeling. Mice were sacrificed 4 days after treatment with CLR1501 (16 mg/kg IV). Fluorescence of tumor versus contralateral brain tissue was quantified with flow cytometry. Samples were also cryosectioned, nuclear counterstained, immunolabeled with human specific antibodies, and evaluated with confocal microscopy.
Results: In vitro CLR1501 fluorescence intensity was significantly higher for tumor than NHA in both GSC and GBM cell lines (range tumor/normal ratio = 2.23-4.87). With in vivo CLR1501 administration, fluorescence intensity was significantly higher in tumor than contralateral normal brain (tumor/normal ratio of 2.74). The tumor border was clearly visible using CLR1501 fluorescence with confocal imaging of cryosections (tumor/normal ratio of 7.85 and 2.51). Strong CLR1501 signal persists in vitro 2-3 weeks after re-isolation of in vivo treated GSC xenografts.
Conclusions: CLR1501 is a novel fluorescent cellular imaging agent that is preferentially retained in GBM. This is the first demonstration that CLR1501 avidly labels the entire GBM tumor (including GSCs) with long-term retention. Tumor cell selectivity suggests a potential for fluorescence-guided surgery. Improved fluorescent tumor markers are needed since the most commonly used agent, 5-aminolevulinic acid (5-ALA), does not visibly label all gliomas. We are carrying out ongoing preclinical and clinical studies with CLR1501-related molecules for GBM and other cancers.
Patient Care: Intraoperative fluorescence guidance enhances the ability to identify tumor cells and increases the extent of tumor resection, which is one of the determinants of tumor patient outcome. In this study we describe a novel fluorescent agent, CLR1501, which is selectively retained in cancer, including GBM, and has the potential for use in fluorescence-guided surgery.
Learning Objectives: By the conclusion of this session, participants should be able to: 1) Understand the tumor labeling capabilities of the novel fluorescent agent CLR1501 and 2) Discuss the potential role of fluorescence-guided surgery to increase extent of tumor resection.
References: 1. Stummer W, Pichlmeier U, Meinel T, et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 2006;7(5):392–401.
2. Valdes PA, Kim A, Brantsch M, et al. delta-aminolevulinic acid-induced protoporphyrin IX concentration correlates with histopathologic markers of malignancy in human gliomas: the need for quantitative fluorescence-guided resection to identify regions of increasing malignancy. Neuro-oncology. 2011;13(8):846–856