Development, assessment and in vivo results of a novel ultrasound imaging technique for lymph node staging in a mouse model of metastasis
Cancer is the second leading cause of death worldwide, responsible for 1 in 6 deaths in 2018 (9.6 million deaths)(World Health Organization, 2017). Early detection is of upmost importance to increase patients’ chances of recovery, improve survival rates and reduce the adverse side effects triggered by alternative treatment procedures (major surgical resections, aggressive radiotherapy and/or chemotherapy) (Etzioni et al., 2003; Gegechkori, Haines and Lin, 2017; Nurgali, Jagoe and Abalo, 2018; Dilalla et al., 2020). Various medical imaging techniques such as MRI, PET and SPECT and ultrasound (US) are routinely used for detection and treatment planning, however the lack of sufficient spatial resolution and contrast on clinical images does not provide satisfactory lymph node (LN) assessment; despite the nodes’ involvement in the metastatic process and incorporation in the TNM (tumour, node, metastasis) staging system (Edge et al., 2010). US is the second most common imaging technique performed in hospital after radiography, and is widely available. The European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) have encouraged development of US techniques for more accurate characterisation of LN involvement in disease. This has led to the development of pre-clinical magneto-motive ultrasound (MMUS) imaging techniques, using nano-sized paramagnetic nanoparticles (SPIONs), to delineate and provide information on the LN’s stiffness by assessing the contrast agent displacement (Bruno et al., 2015; Sjöstrand, Evertsson and Jansson, 2020). The addition of SPIONs to clinically used microbubble (MBs) contrast agents was then proposed for the novel development of a contrast enhanced (CE) MMUS (CE-MMUS) technique to improve the sensitivity of the existing MMUS technique. Importantly, in silico finite element analysis (FEA) have predicted that the CE-MMUS would induce a 2.3x larger displacement than MMUS (Sjöstrand et al., 2019). This project aimed to develop, test and compare the novel CE-MMUS technique with MMUS. The work was carried out on phantom models and in vivo on healthy and tumour-bearing mice harbouring LN metastases. To do so, a multidisciplinary team of which I was a part, replicated a MMUS setup in Glasgow/Edinburgh; produced tissue mimicking materials; produced novel SPION-microbubble (MB) contrast agents; created mouse models of metastasis appropriate for the development of CE-MMUS; and performed CE-MMUS and MMUS in silico, ex vivo and in vivo. Intestinal organoids issued from Shroom2KO and KPN mouse models were injected into healthy C57BL/6J animals via different routes, to assess their metastatic potential for developing tumours for detection with US. Whilst injection of intestinal Shroom2KO or C57BL/6J (control) intestinal organoids did not induce any detectable tumour or metastasis after 30 weeks, KPN intestinal tumour organoids via hock injection induced leg growths and detectable leg-draining LN metastases in 86.6% and 53.9% respectively of recipient mice from 4 weeks post-injection. Due to a lack of data on murine LNs in the literature, LNs of interest for our project were studied via dissection, histology and various US techniques (B-mode, 3D, contrast). 100% inguinal LNs draining a tumour-bearing leg were considerably larger than inguinal LNs draining a tumour-free leg. 3D-US performed at endpoint on five animals indicate that the tumour-draining inguinal LNs were on average 8.4 times larger than the tumour-free ones. 3D-US also indicated that the tumour-draining LNs were changing shape, becoming rounder, and CEUS experiments showed that the blood flow in tumour-draining LNs was reduced by up to 70%. After performing post-mortem dissection of the LNs to assess the presence of metastases, this pilot experiment revealed that the measurement of several analytical parameters increased the sensitivity of LN metastatic detection compared to size only – size currently being the only parameter utilised clinically to determine LN metastasis (Bacou et al., 2022). To develop and test the CE-MMUS imaging technique, the engineers first fabricated an electromagnet and combined it to the Vevo3100 ultrasound system to create the MMUS setup. PVA-based SPION-loaded tissue mimicking materials (TMM) were fabricated to imitate LNs and test the MMUS imaging technique before using it in vivo. These TMM studies showed promising results. Biocompatible SPION-MBs were produced for CE-MMUS in vivo experiments by conjugating dextran-coated paramagnetic oxide particles to biotin and linking them to Target-Ready MicroMarker®. Target-Ready MicroMarker® is a commercially available preclinical contrast agent consisting in lyophilised lipids bound to streptavidin, that can easily be resuspended into microbubbles and used for in vivo studies. Pilot MMUS and CE-MMUS experiments were performed on three tumour-free C57BL/6J animals. Animals were injected with 35g biotinylated SPIONs into the hock 24h prior to experiments, then scanned using MMUS and CE-MMUS (post MicroMarker® injection) whilst under terminal anaesthesia. Preliminary results showed that the injection of targeted MicroMarker® (CE-MMUS) in addition to SPIONs induced a larger and more easily detectable signal in the same assessed LNs than SPIONs alone (0.47±0.03m and 0.39±0.05m respectively). In conclusion, a MMUS setup, bimodal SPION-MBs contrast agents, and a mouse model of metastasis were successfully created for the project. Those various experiments generated the first proof of principle that the combination of SPION-MBs (CE-MMUS) induce a larger mean displacement than SPIONs only (MMUS). Going forward, greater numbers of tumour-free as well as tumour-bearing mice will be needed to further develop and improve the CE-MMUS data acquisition and analysis, however these preliminary CE-MMUS experiments in vivo show the potential of this novel technique in offering accurate characterisation of LNs in cancer imaging.