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

Contrast-enhanced magnetomotive ultrasound imaging (CE-MMUS) for colorectal cancer staging : assessment of sensitivity and resolution to detect alterations in tissue stiffness

A key challenge in the treatment of colorectal cancer is identification of the sentinel draining lymph node. Magnetomotive ultrasound, MMUS, has identified lymph nodes in rat models: superparamagnetic iron oxide nanoparticles (SPIONs) accumulated in the lymph are forced to oscillate by an external magnetic field; the resulting axial displacement is recovered allowing structure delineation with potential to indicate alterations in tissue stiffness, but it is limited by small vibration amplitudes. We propose CE-MMUS using SPION loaded microbubbles (SPION-MBs) to enhance sensitivity, reduce toxicity, and offer additional diagnostic or perfusion information. Laser doppler vibrometry measurements was performed on SPION containing tissue mimicking material during magnetic excitation. These measurements show a vibration amplitude of 279 ± 113 μm in a material with Young's modulus of 24.3 ± 2.8 kPa, while the displacements were substantially larger, 426 ± 9 μm, in the softer material, with a Young's modulus of 9.6 ± 0.8 kPa. Magnetic field measurement data was used to calibrate finite element modelling of both MMUS and CE-MMUS. SPION-MBs were shown to be capable of inducing larger tissue displacements under a given magnetic field than SPIONs alone, leading to axial displacements of up to 2.3x larger. A doubling in tissue stiffness (as may occur in cancer) reduces the vibration amplitude. Thus, there is potential for CE-MMUS to achieve improved stiffness sensitivity. Our aim is to define the potential contribution of CE-MMUS in colorectal cancer diagnosis and surgical guidance

Development of Preclinical Ultrasound Imaging Techniques to Identify and Image Sentinel Lymph Nodes in a Cancerous Animal Model

Lymph nodes (LNs) are believed to be the first organs targeted by colorectal cancer cells detached from a primary solid tumor because of their role in draining interstitial fluids. Better detection and assessment of these organs have the potential to help clinicians in stratification and designing optimal design of oncological treatments for each patient. Whilst highly valuable for the detection of primary tumors, CT and MRI remain limited for the characterization of LNs. B-mode ultrasound (US) and contrast-enhanced ultrasound (CEUS) can improve the detection of LNs and could provide critical complementary information to MRI and CT scans; however, the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) guidelines advise that further evidence is required before US or CEUS can be recommended for clinical use. Moreover, knowledge of the lymphatic system and LNs is relatively limited, especially in preclinical models. In this pilot study, we have created a mouse model of metastatic cancer and utilized 3D high-frequency ultrasound to assess the volume, shape, and absence of hilum, along with CEUS to assess the flow dynamics of tumor-free and tumor-bearing LNs in vivo. The aforementioned parameters were used to create a scoring system to predict the likelihood of a disease-involved LN before establishing post-mortem diagnosis with histopathology. Preliminary results suggest that a sum score of parameters may provide a more accurate diagnosis than the LN size, the single parameter currently used to predict the involvement of an LN in disease

Contrast enhanced magneto-motive ultrasound in lymph nodes - modelling and pre-clinical imaging using magnetic microbubbles

Despite advances in MRI, the detection and characterisation of lymph nodes in rectal cancer remains complex, especially when assessing the response to neo-adjuvant treatment. An alternative approach is functional imaging, previously shown to aid characterization of cancer tissues. We report proof-of-concept of the novel technique Contrast-Enhanced Magneto-Motive Ultrasound (CE-MMUS) to recover information relating to local perfusion and lymphatic drainage, and interrogate tissue mechanical properties through magnetically induced tissue deformations. The feasibility of the proposed application was explored using a combination of pre-clinical ultrasound imaging and finite element analysis. First, contrast enhanced ultrasound imaging on one wild type mouse recorded lymphatic drainage of magnetic microbubbles after bolus injection. Second, preliminary CE-MMUS data were acquired as a proof of concept. Third, the magneto-mechanical interactions of a magnetic microbubble with an elastic solid were simulated using finite element software. Accumulation of magnetic microbubbles in the inguinal lymph node was verified using contrast enhanced ultrasound, with peak enhancement occurring 3.7 s post-injection. Preliminary CE-MMUS indicates the presence of magnetic contrast agent in the lymph node. The finite element analysis explores how the magnetic force is transferred to motion of the solid, which depends on elasticity and bubble radius, indicating an inverse relation with displacement. Combining magnetic microbubbles with MMUS could harness the advantages of both techniques, to provide perfusion information, robust lymph node delineation and characterisation based on mechanical properties. Clinical Relevance— Robust detection and characterisation of lymph nodes could be aided by visualising lymphatic drainage of magnetic microbubbles using contrast enhanced ultrasound imaging and magneto-motion, which is dependent on tissue mechanical properties

Contrast enhanced magneto-motive ultrasound in lymph nodes - modelling and pre-clinical imaging using magnetic microbubbles

Despite advances in MRI, the detection and characterisation of lymph nodes in rectal cancer remains complex, especially when assessing the response to neo-adjuvant treatment. An alternative approach is functional imaging, previously shown to aid characterization of cancer tissues. We report proof-of-concept of the novel technique Contrast-Enhanced Magneto-Motive Ultrasound (CE-MMUS) to recover information relating to local perfusion and lymphatic drainage, and interrogate tissue mechanical properties through magnetically induced tissue deformations. The feasibility of the proposed application was explored using a combination of pre-clinical ultrasound imaging and finite element analysis. First, contrast enhanced ultrasound imaging on one wild type mouse recorded lymphatic drainage of magnetic microbubbles after bolus injection. Second, preliminary CE-MMUS data were acquired as a proof of concept. Third, the magneto-mechanical interactions of a magnetic microbubble with an elastic solid were simulated using finite element software. Accumulation of magnetic microbubbles in the inguinal lymph node was verified using contrast enhanced ultrasound, with peak enhancement occurring 3.7 s post-injection. Preliminary CE-MMUS indicates the presence of magnetic contrast agent in the lymph node. The finite element analysis explores how the magnetic force is transferred to motion of the solid, which depends on elasticity and bubble radius, indicating an inverse relation with displacement. Combining magnetic microbubbles with MMUS could harness the advantages of both techniques, to provide perfusion information, robust lymph node delineation and characterisation based on mechanical properties. Clinical Relevance— Robust detection and characterisation of lymph nodes could be aided by visualising lymphatic drainage of magnetic microbubbles using contrast enhanced ultrasound imaging and magneto-motion, which is dependent on tissue mechanical properties

Development of Preclinical Ultrasound Imaging Techniques to Identify and Image Sentinel Lymph Nodes in a Cancerous Animal Model

Lymph nodes (LNs) are believed to be the first organs targeted by colorectal cancer cells detached from a primary solid tumor because of their role in draining interstitial fluids. Better detection and assessment of these organs have the potential to help clinicians in stratification and designing optimal design of oncological treatments for each patient. Whilst highly valuable for the detection of primary tumors, CT and MRI remain limited for the characterization of LNs. B-mode ultrasound (US) and contrast-enhanced ultrasound (CEUS) can improve the detection of LNs and could provide critical complementary information to MRI and CT scans; however, the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) guidelines advise that further evidence is required before US or CEUS can be recommended for clinical use. Moreover, knowledge of the lymphatic system and LNs is relatively limited, especially in preclinical models. In this pilot study, we have created a mouse model of metastatic cancer and utilized 3D high-frequency ultrasound to assess the volume, shape, and absence of hilum, along with CEUS to assess the flow dynamics of tumor-free and tumor-bearing LNs in vivo. The aforementioned parameters were used to create a scoring system to predict the likelihood of a disease-involved LN before establishing post-mortem diagnosis with histopathology. Preliminary results suggest that a sum score of parameters may provide a more accurate diagnosis than the LN size, the single parameter currently used to predict the involvement of an LN in disease

DDR1 and DDR2 physical interaction leads to signaling interconnection but with possible distinct functions

<p>Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are members of the tyrosine kinase receptors activated after binding with collagen. DDRs are implicated in numerous physiological and pathological functions such as proliferation, adhesion and migration. Little is known about the expression of the two receptors in normal and cancer cells and most of studies focus only on one receptor. Western blot analysis of DDR1 and DDR2 expression in different tumor cell lines shows an absence of high co-expression of the two receptors suggesting a deleterious effect of their presence at high amount. To study the consequences of high DDR1 and DDR2 co-expression in cells, we over-express the two receptors in HEK 293T cells and compare biological effects to HEK cells over-expressing DDR1 or DDR2. To distinguish between the intracellular dependent and independent activities of the two receptors we over-express an intracellular truncated dominant-negative DDR1 or DDR2 protein (DDR1DN and DDR2DN). No major differences of Erk or Jak2 activation are found after collagen I stimulation, nevertheless Erk activation is higher in cells co-expressing DDR1 and DDR2. DDR1 increases cell proliferation but co-expression of DDR1 and DDR2 is inhibitory. DDR1 but not DDR2 is implicated in cell adhesion to a collagen I matrix. DDR1, and DDR1 and DDR2 co-expression inhibit cell migration. Moreover a DDR1/DDR2 physical interaction is found by co-immunoprecipitation assays. Taken together, our results show a deleterious effect of high co-expression of DDR1 and DDR2 and a physical interaction between the two receptors.</p

Modelling of magnetic microbubbles to evaluate contrast enhanced magneto-motive ultrasound in lymph nodes – a pre-clinical study

Objectives Despite advances in MRI the detection and characterisation of lymph nodes in rectal cancer remains complex, especially when assessing the response to neo-adjuvant treatment. An alternative approach is functional imaging, previously shown to aid characterization of cancer tissues. We report proof of concept of the novel technique Contrast-Enhanced Magneto-Motive Ultrasound (CE-MMUS) to recover information relating to local perfusion and lymphatic drainage, and interrogate tissue mechanical properties through magnetically induced deformations. Methods The feasibility of the proposed application was explored using a combination of experimental animal and phantom ultrasound imaging, along with finite element analysis. First, contrast enhanced ultrasound imaging on one wild type mouse recorded lymphatic drainage of magnetic microbubbles after bolus injection. Second, tissue phantoms were imaged using MMUS to illustrate the force- and elasticity dependence of the magneto-motion. Third, the magneto-mechanical interactions of a magnetic microbubble with an elastic solid were simulated using finite element software. Results Accumulation of magnetic microbubbles in the inguinal lymph node was verified using contrast enhanced ultrasound, with peak enhancement occurring 3.7 s post injection. The magnetic microbubble gave rise to displacements depending on force, elasticity, and bubble radius, indicating an inverse relation between displacement and the latter two. Conclusions Combining magnetic microbubbles with MMUS could harness the advantages of both techniques, to provide perfusion information, robust lymph node delineation and characterisation based on mechanical properties. Advances in knowledge (a) Lymphatic drainage of magnetic microbubbles visualised using contrast enhanced ultrasound imaging and (b) magneto-mechanical interactions between such bubbles and surrounding tissue could both contribute to (c) robust detection and characterisation of lymph nodes

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy

International audienceTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer