Bone sarcomas are devastating primary bone cancers that mostly affect children, young adults, and the elderly. These aggressive tumors are associated with poor survival, and surgery remains the mainstay of treatment. Surgical planning is increasingly informed by positron emission tomography (PET), and tumor margin identification during surgery is aided by near-infrared fluorescence (NIRF) imaging, yet these investigations are confounded by probes that lack specificity for sarcoma biomarkers. We report the development of a dual-modal (PET/NIRF) immunoconjugate ([ 89 Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW) that targets MT1-MMP, a matrix metalloproteinase overexpressed in highgrade sarcomas. [ 89 Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW was synthesized via site-specific chemoenzymatic glycan modification, characterized, and isolated in high specific activity and radiochemical purity. Saturation binding and immunoreactivity assays indicated only minor perturbation of binding properties. A novel mouse model of dedifferentiated chondrosarcoma based on intrafemoral inoculation of HT1080 WT or KO cells (high and low MT1-MMP expression, respectively) was used to evaluate target binding and biodistribution. Fluorescence and Cerenkov luminescence images of [ 89 Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW showed preferential uptake in HT1080 WT tumors. Ex vivo gamma counting revealed that uptake in MT1-MMP-positive tumors was significantly higher than that in control groups. Taken together, [ 89 Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW is a promising dual-modal sarcoma imaging agent for pre-operative surgical planning and intraoperative surgical guidance.

Introduction: Indocyanine green (ICG) is a near infrared (NIR) dye which has been used clinically for over 50 years and has recently been utilised for fluorescence guided surgery in a number of cancer types, including sarcoma. ICG is taken up and retained by sarcoma tumours to a greater extent than normal tissue, demonstrating its potential to aid in visualisation of tumour margins. However, the mechanisms surrounding preferential ICG uptake in tumours are poorly understood. Methods: In vitro ICG cellular uptake studies were performed across a panel of four sarcoma cell lines and one breast cancer cell line, exhibiting varying proliferation rates and phenotypes. The effects of ICG concentration, incubation time, inhibition of clathrin mediated endocytosis and cell line proliferation rate on the cellular uptake of ICG were investigated, using fluorescence microscopy and flow cytometry. The spatial orientation of ICG was also assessed in a patient specimen. Results: The level of ICG cellular uptake was dependent on ICG concentration and incubation time. Cell line proliferation rate correlated significantly to ICG uptake within 30 minutes (Rs = 1, p<0.001), whilst retention of ICG after 24hrs did not (Rs = 0.3, p=0.624). From our data, the primary mechanism of ICG uptake in sarcoma cells is via clathrin mediated endocytosis. Following the resection of a grade 3 leiomyosarcoma, ICG signal was detectable macroscopically and on 3um sections, whilst being negative on the muscle control. Conclusions: The use of ICG for tumour detection in sarcoma surgery may demonstrate higher utility in high grade tumours compared to low grade tumours, due to the observation of higher ICG uptake in more proliferative cell lines. It is likely that the enhanced permeability and retention (EPR) effect plays a significant role in the retention of ICG within tumours. Future work on the detection of ICG at the cellular level within human tissue sections is required, with the aid of purpose built NIR microscopes.

A systematic investigation into the influence of degree of labelling of antibody-cell penetrating peptide conjugates upon cellular internalisation.