Ida Thorøe Michler is a science student enrolled at the MSc in Human Biology at The University of Copenhagen. She is doing a nine month Master’s thesis project (50 ECTS) at Minerva Imaging. This is a joint project supervised by Sigrid Cold and Carsten Haagen Nielsen at Minerva Imaging and Prof. Andreas Kjær at Cluster for Molecular Imaging (BMI) at UCPH.
The aim of this Master’s project is to generate immortalized cell lines from patient derived xenograft (PDX) models of glioblastoma and breast cancer origin. PDX models are initiated by transplanting tumor pieces obtained from patients directly into mice without the need for in vitro cultivation. While PDX models closely represent the heterogeneity and drug response of patient tumors, they are costly, require extra mice for expansion steps, and take a long time to grow. Replacing these models with PDX derived immortalized cell lines will provide a cost-effective, faster and more translatable tool for conducting pre-clinical studies in vivo and in vitro, while also reducing animal usage in line with 3Rs and good animal welfare practices.
Cells from subcutaneous PDX tumors will be isolated and cultivated to generate immortal cell lines. Once established, the viability and growth potential of these cell lines will be evaluated after multiple thaw- and freeze cycles to ensure their suitability for long term use. The cell lines will be characterised by using flow cytometry to determine the ratio of human/mouse cells after murine depletion and to quantify relevant target proteins, confirming that the identity of the immortalized cells matches that of the original PDX.
Once characterized, tumor-formation ability will be investigated by inoculating cells either subcutaneously or orthotopically (at the site of origin) in immunodeficient mice and monitoring tumor growth by external calliper measurements or T2*-weighted magnetic resonance imaging (MRI). Similarities between the original PDX models and the PDX-derived cell lines will be investigated with immunohistochemistry. Cell lines will be transfected with the luciferase gene and cell growth and viability after anti-cancer treatments will be measured by bioluminescence. This also allows tumor growth and metastasis to be monitored using bioluminescence instead of the more time-consuming MRI. Finally, qPCR will be used to evaluate methylation status as an indication for cell line resistance to chemotherapy.
Signe Svenning Grønager is a vet student enrolled in the MSc in Veterinary Medicine at The University of Copenhagen. She is doing a nine month long Master’s thesis project (30 ECTS) at Minerva Imaging.
Michala Nordfalk is a science student enrolled in the MSc in Human Biology at The University of Copenhagen. She is doing a nine month long Master’s thesis project (50 ECTS) at Minerva Imaging.
Signe and Michala are both co-supervised by Mette Flethøj Madsen at Minerva Imaging and Prof. Andreas Kjær at The Department of Clinical Physiology, Nuclear Medicine & PET at Rigshospitalet and Cluster for Molecular Imaging at The University of Copenhagen.
Myocardial Infarction (MI), also called heart attack, is when a blockage within the coronary artery reduces or stops blood flow to the heart muscle and causes muscle damage due to lack of oxygen. Acute myocardial infarction affects more than 3 million people globally per year and is a leading cause of death in the Western world (1). Rat MI models are often used for long-term pharmacological studies to investigate recovery processes of the infarcted area. In these rats, MI is currently induced by open thorax surgery with either transient occlusion (30-60 minutes) or permanent ligation of the left anterior descending (LAD) artery (2,3). These models are quite invasive, have high variation in infarction size, and are associated with a high mortality rate of 15-38% (4–6).
Signe and Michala will work together to establish and validate a new minimally invasive MI model in rats using ultrasound-guided electrocoagulation of the LAD artery. This method allows permanent blockage of the coronary artery without open surgery, providing the advantage that intubation and thoracotomy are avoided to improve the overall welfare of the animals. Once the surgical methods are established, each student will continue with separate projects to validate and characterise the new model according to their scientific backgrounds and interests. This collaboration will provide Minerva Imaging with new imaging workflows, lab methods, data analysis, and related protocols for our commercial activities within the cardiac space.
The establishment of the minimally invasive model will be compared to permanent LAD ligation one and five weeks after induction using current gold standard MRI methods to measure infarct size and ejection fraction. In addition, angiogenesis and repair within the damaged tissue will be quantified by arginine-glycine-aspartic acid (RDG)-PET/CT and tissue fibrosis will be quantified with late gadolinium enhancement MRI. For further characterization of the model, blood samples will be collected 24 hours post-operation for ELISA quantification of troponin (tissue damage biomarker (7)) and heart samples collected at study end will be stained with the redox indicator, triphenyltetrazolium chloride (TTC) to confirm infarct size ex vivo. The heart samples will then be used for immunohistochemical detection of the angiogenesis-marker, integrin αvβ3.
The minimally invasive model will be compared to traditional permanent LAD ligation by evaluation of cardiac perfusion since reduced perfusion within the heart correlates with the infarct size and location in vivo. Cardiac perfusion will be measured with fluorodeoxyglucose (FDG)-PET/CT and Technetium 99m sestamibi (MIBI)-SPECT/CT imaging one week post-MI induction. An animal welfare scoring system specific for this experimental model will be established and compared with infarct size and other outcomes. The animals will be closely monitored to identify and treat any complications as well as monitoring pain levels and responding with appropriate pharmacological and veterinary care.
Figure: Myocardial Infarction project overview, timeline created using BioRender.
- Global Awareness of Myocardial Infarction Symptoms in General Population. Korean Circ J. 2021; 51(12): 997–1000.
- Wang et al. Self-Gated Late Gadolinium Enhancement at 7T to Image Rats with Reperfused Acute Myocardial Infarction. Korean J Radiol. 2018; 19: 247–255.
- Martin et al. Preclinical Models of Myocardial Infarction: From Mechanism to Translation. Br J Pharmacol. 2022; 179: 770–791.
- Waller et al Serial Magnetic Resonance Imaging of Microvascular Remodeling in the Infarcted Rat Heart; 2001; 103:1564-1569
- Bentsen et al. Myocardial Perfusion Recovery Induced by an α-Calcitonin Gene-Related Peptide Analogue. Journal of Nuclear Cardiology. 2022; 29: 2090–2099.
- Gao et al. PET Imaging of Angiogenesis after Myocardial Infarction/Reperfusion Using a One-Step Labeled Integrin-Targeted Tracer 18F-AlF-NOTA-PRGD2. Eur J Nucl Med Mol Imaging. 2012; 39: 683–692.
- Frobert et al. Prognostic Value of Troponin I for Infarct Size to Improve Preclinical Myocardial Infarction Small Animal Models. Front Physiol. 2015; 6: 353.
Sarah Wilkens Wulff is a vet student enrolled in the MSc in Veterinary Medicine at the University of Copenhagen. She has successfully completed a 6 month masters thesis project at Minerva Imaging (30 ECTS points). This was a joint project supervised by Philip G. J. Pedersen at Minerva Imaging and Prof. Andreas Kjær at the Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Department of Biomedical Sciences, Rigshospitalet and University of Copenhagen.
Nonalcoholic Steatohepatitis (NASH) is a lifestyle disease where fat accumulation in the liver causes inflammation, fibrosis and reduced liver function (1). NASH is a serious condition that can progress to more severe liver disease, such as cirrhosis, liver failure, and increased risk of liver cancer. It is a growing health concern worldwide, especially in developed countries where obesity and metabolic disorders are prevalent.
NASH is challenging to diagnose as only patients with advanced fibrosis show clinical symptoms. Today the only reliable diagnostic tool is to obtain a liver biopsy for histopathology which is an invasive and potentially risky procedure (2). To avoid this, extensive work has been done to find and validate diagnostic biomarkers or imaging techniques but still none of these efforts have proven to be sufficiently reliable.
Different Fibroblast activation protein inhibitors (FAPI) are currently being investigated as targets for diagnostic imaging and therapy within both oncology and fibrotic liver diseases (3-5).
The aim of this Master’s project was to test 68Ga-FAPI PET/CT imaging as a diagnostic tool to quantify liver size, density and liver fibrosis in preclinical rat models. NASH was induced in the rats either by feeding a choline-deficient L-amino acid defined (CDAA) high fat diet (HFD) for 12 weeks, after which time they developed liver steatosis (or abnormal fat accumulation), fibrosis and inflammation (6), or by surgical bile duct ligation, a consistent and repeatable model of liver fibrosis (7).
The result of the study showed that CDAA-HFD fed rats had a significantly higher uptake of 68Ga-FAPI PET/CT compared to controls indicating the presence of liver fibrosis in these animals. Furthermore, CT image analysis showed that initial liver steatosis reduced liver density followed by a gradual increase in liver density as fibrosis developed, similarly to NASH progression in human patients (8). Fibrosis infiltration of the liver was confirmed using histopathology of tumor biopsies (Sirius red area fraction). As in the CDAA model, a significantly higher uptake of 68Ga-FAPI PET/CT was also seen in rats four weeks after bile duct ligation when compared to SHAM operated controls.
We conclude that non-invasive 68Ga-FAPI PET/CT imaging has the potential to precisely visualize and measure liver fibrosis and liver density in pre-clinical models of NASH and therefore has potential as a diagnostic tool in the clinic for the benefit of patients.
Figure: A) 68Ga-FAPI liver uptake by PET imaging at baseline and after 12 wks. of CDAA-HFD presented as standard uptake value (SUV). B) Sirius red (SR) staining of the liver. Analysed using Visiopharm. All graphs: mean ± SD, n=5-9. ***p<0.001 and ****p<0.0001, by two-tailed t-test.
- Hardy et al. Nonalcoholic Fatty Liver Disease: Pathogenesis and Disease Spectrum. Annu Rev Pathol. 2016; 11:451-96. DOI: 10.1146/annurev-pathol-012615-044224
- Meyer C et al. Radiation Dosimetry and Biodistribution of 68Ga-FAPI-46 PET Imaging in Cancer Patients. J Nucl Med. 2020 Aug;61(8):1171-1177. doi: 10.2967/jnumed.119.236786.
- Pirasteh A et al. Staging Liver Fibrosis by Fibroblast Activation Protein Inhibitor PET in a Human-Sized Swine Model. J Nucl Med. 2022 Dec;63(12):1956-1961. doi: 10.2967/jnumed.121.263736.
- Wang XL, Li H, Wang QS, Zhang XL. Clinical value of pre-and postoperative 18F-FDG PET/CT in patients undergoing liver transplantation for hepatocellular carcinoma. Nan Fang Yi Ke Da Xue Xue Bao. 2006 Aug;26(8):1087-91, 1095. PMID: 16939890.
- Xiaohan Fang, Man Xie, Youwei Zhao et al. Clinical Value of 18F-FAPI PET/CT in assessing early-stage fibrosis of graft after liver transplantation: preliminary experience, 05 October 2022, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-2092805/v1]
- Carreres et al. Modeling Diet-Induced NAFLD and NASH in Rats: A Comprehensive Review. Biomedicines. 2021; 9(4): 378. DOI: 10.3390/biomedicines9040378
- Kirkland et al. Reversible surgical model of biliary inflammation and obstructive jaundice in mice. J. Surg. Res. 2010; 164: 221–227. DOI: 10.1016/j.jss.2009.08.010
- Tisch et al. Hounsfield unit values of liver pathologies in unenhanced post-mortem computed tomography. Int J Legal Med. 2019; 133(6): 1861-1867. DOI: 10.1007/s00414-019-02016-1