Multimodal PET yields novel insights in preclinical oncology

By Mette Lauritzen PhD, Product Marketing Manager, Nuclear Molecular Imaging, Bruker BioSpin

Different types of tumours, and their varying reactions to treatment, make the search for new effective cancer therapies incredibly challenging. Preclinical in vivo imaging methods are helping to obtain further knowledge of tumour morphology, progression, and biomarker expression by enabling the visualisation of cancer-related processes in real-time. Non-invasive in vivo imaging technologies such as positron emission tomography (PET) are helping researchers better understand the course of tumour progression – and gain more knowledge of how to treat them.

Molecular heterogeneity, e.g., the variability among cancer cells, within a single tumour poses a considerable challenge in cancer therapy. Imaging, together with better understanding of cancer genomics and developments in molecular pathology, are key players in achieving personalised cancer treatment. These factors are driving advances in preclinical PET oncology research, with the goal of providing patients with more precise drugs and drug delivery systems.

The move from pure anatomical imaging to molecular imaging with PET has enabled research­ers to visualise tumour heterogeneity, which is especially impor­tant with regards to administering combination therapies for cancer. Over the past 15 years, the expansive development of imaging technologies has moved beyond a simple anatomical approach towards more complex multimodal systems. Such systems are expected to become the standard of care within the next few years.

Multimodal imaging, in parallel with genomic profiling, could allow for visualisation of drug-induced changes in a specific biochemical process. As a result, researchers could deliver insights into drug target engagement or alterations in tumour phenotype.

Multimodal imaging

PET provides three-dimensional (3D) functional imaging using radioactive tracers (radiotracers), showing the spatial distribution of biomolecular activity in the bodies of animal models and humans. When PET is combined with other imaging modalities – such as magnetic resonance imaging (MRI), computed tomogra­phy (CT), and single-photon emission computed tomography (SPECT), researchers are able to link structural and functional imaging in one experi­ment.

Hundreds of PET radiotracers have been developed and evaluated for preclinical oncology research, with dozens of Food and Drug Administration (FDA)-approved radiotracers cur­rently available.1

Use of multimodal imaging in preclinical oncology research

Small animal imaging deepens our understanding of disease development and the effect of potential treatments in the preclinical phase, and advances in PET technology are powering the translation of this research into the clinical setting. Preclinical imaging provides important insights into dis­ease mechanisms, from molecular to organ level, and contributes to the development and evaluation of novel therapeutic strategies. In this con­text, researchers can incorporate PET imaging in the drug development phase to provide data that can be extrapolated from animal to human studies.

Combining PET with the soft tissue morphological imaging from MRI or anatomical information provided by CT scanning are gaining popularity in preclinical imaging applications. PET/CT is a valuable tool in oncol­ogy research due to its ease-of-use, high-throughput capabilities, and high resolution for bone and pulmonary applications. Since the mid-1990s, researchers have used PET/CT to study cancer therapeutics and tumour biology, and for tracer development.

PET/MR, while less established in preclinical imaging, is gaining ground in oncology due to its ability to image with lower ionising radiation, and its potential for multipara­metric imaging. Another benefit is MRI’s superior anatomical soft tissue contrast, which offers the ability to detect tumour margins, evaluate tracer distribu­tion within individual tumours to generate volume of inter­est (VOI), and calculate standardised uptake value (SUV) in a range of preclinical models – thereby improving the functional analysis of complementary PET data.

Whereas PET and CT data are acquired consecutively, PET/MR sys­tems can acquire data simultaneously, optimising imaging workflows. The combination of PET and MRI technology can be applied to many processes in tumour biology, such as the role of tumour microenvironment in tumour progres­sion, and used to simultaneously investigate upstream, downstream, and parallel pathways of metabolism to fully characterise these changes and underlying biology.

Tumour biology analysis

PET can provide information on the expres­sion of receptors, energy metabolism, and other biomarkers of tumours, by imaging an intravenously injected radiotracer – a radioisotope attached to a molecular probe that targets a specific molecule or metabolic pathway – or uptake by tumour cells.

A key characteristic of tumour cells is their elevated metabolic turno­ver. 18F-fludeoxyglucose (18F-FDG) is often used as a radi­olabeled glucose analogue tracer to analyse glucose uptake in tumours to track their progression and monitor aggressiveness. Commonly used PET tracers like 18F-FDG or 18F-Fluoro­thymidine (FLT) can moni­tor universal markers of tumour physiology, including altered metabolism and proliferation.

More specific PET agents can target the expression of one molecule or gene product, therefore potentially helping researchers better understand and assess tumour biology and therapy responses. The PET radiopharmaceutical 68Ga-PSMA has revolutionised prostate cancer imaging in recent years. The prostate-specific membrane antigen (PSMA) found in cell membranes is highly expressed in the prostate and in prostate cancer cells, making 68Ga-PSMA very effective in imaging. This tracer has the added advantage of using the PET isotope gallium-68 (Ga-68), which has comparatively lower production costs.

Tumour vascularisation and blood flow is another indicator of tumour biology. Its visualisation requires radiotracers than can diffuse freely throughout the vascular system and across tumour cell membranes, such as 15O-water, 13N-ammonia, and 82Rb-chloride2. Chemotherapy can be used to target the tumour’s microvascular system, a therapy which intravenous PET radiotracers can help monitor and optimise.

Theranostics

Theranostics inte­grates diagnosis and therapeutics, and it represents a powerful step towards more personalised treatment strategies. Recent developments in molecular biology, proteomics and genetics have significantly enriched understanding of tumour biochemis­try and function, including identification of the receptors that tumour cells express. In theranostics, these molecular targets can be used to access tumours, image the disease area, and deliver targeted cytotoxic substances directly to tumour tissue.

In PET and SPECT imaging applications, molecular targets can be combined with radiotracers for both diagnostic and therapeu­tic purposes. Diagnostic imaging can determine tumour size, classification and stage. As a result, localised radiation can be administered specifically to the diseased area, without damaging surrounding healthy cells.

A lack of affordable radiotracers has prevented widespread uptake of theranostics and the approach is currently limited. However, the development of new radiotracers aims to free up theranostics as a tool for many other types of cancer.

Folate-based radiopharmaceuticals

The folate receptor had limited expres­sion in healthy cells, but is over expressed in a variety of highly proliferating cancer cells, particularly ovarian and endometrial cancers, making it a key target for detecting and evaluating tumours in vivo. Certain properties, including folate’s low molecular weight and non-immunogenicity, make this type of B vitamin an ideal target for cancer imaging. Many radiolabeled folates have been developed for this purpose, particularly for SPECT imaging.3

Folate-based radiopharmaceuticals for PET imaging are under development, but creating radiolabeled folates with high radio­chemical yield, radiochemical purity, and favourable pharmacoki­netics is challenging. Studies on 18F-alakyne folate identify it as a promising candidate for folate receptor PET imaging, but there remains a need to refine its metabolism profile.4

Imaging biomarkers

Researchers at the Institute of Cancer Research, London, are focused on the development and characterisation of imaging biomarkers to inform and guide cancer treatment for individual patients. The group, led by Dr Gabriella Kramer-Marek, is studying predictive imaging biomarkers, and biomark­ers that help to assess drug resistance and tumour response to drugs, with particular interest in the development of theranos­tic agents against receptors from the epidermal growth factor tyrosine kinase receptor (EGFR) family.

The dimerisation of these receptors promotes the activation of downstream sig­nals that initiate and control a wide range of cellular processes, including proliferation, survival and apoptosis. Overexpression of human EGFR receptors (HER) are found in many human malignancies and have facilitated the development of target-specific drugs, some currently in routine clinical practice and others in clinical trials.

The development of Affibody molecules (Affibody Medical AB) combats is an alternative to bulky antibodies as targeting agents. Their small size (~6.5 kDa), simple structure, stability and solubility makes Affibod­ies candidates for in vivo imaging. Several Affibody binders are available, not limited to HER receptors, that have been radiolabeled for PET and SPECT purposes.

In one study, the group used EGFR-specific radioli­gands to measure EGFR expression in mice with head and neck squamous cell cancer (HNSCC), with the aim to define a predictive biomarker to stratify patients for treatment.5 Cetuxi­mab is currently the only approved anti-EGFR mAb used for the treatment of HNSCC. The ability to monitor and assess the drug’s efficacy and any cetuximab-mediated changes in receptor expression could help inform appropriate dosing with anti-EGFR antibodies.

The group used a radiolabeled Affibody molecule (ZEGFR:03115) to non-invasively measure differences in EGFR expression, using a 89Zr-labeled conjugate to assess tumour-to-organ ratios at different time points, and a 18F-labeled analog to measure the response to cetuximab treatment in vivo. To evaluate whether 89Zr-deferoxamine (DFO)- ZEGFR:03115 could distinguish between tumours with varying levels of EGFR expression, mice bearing CAL27 (EGFR +++), Detroit562 (EGFR ++), and MCF7 (EGFR +) xenografts received the radiotracer and were imaged three hours after injection using PET/CT (Albira PET/SPECT/CT, Bruker BioSpin).

The quantified PET imaging data indicated that the highest levels of radioconjugate accumulation were in CAL27 tumours (Figure 1), which correlated with recep­tor expression measured ex vivo by Western Blot and IHC staining.

To monitor response to cetruximab, 18F-aluminium fluo­ride (AlF)-NOTA- ZEGFR:03115 was administered intravenously to mice bearing HN5 tumours (EGFR ++++). The group observed significantly lower uptake in ceuximab-treated mice than in control HN5 tumours (Figure 2).

These results, together with an insignificant change in tumour volume during treatment, highlight the potential for using EGFR imaging as a tool for assessing cetruximab efficacy based on receptor level, rather than relying purely on anatomi­cal imaging, and could provide image-guided therapeutic strategies for the clinic.

Most recently, Dr Kramer-Marek’s group highlighted the need the development of F-18-based imaging biomarkers to monitor therapeutic response in neuroblastoma (NB) patients. Targeted radiotherapy with the noradrenaline analogue, meta-iodobenzylguanidine, radiolabeled with iodine-131 (131I-mIBG), has been widely used as a theranostic pair for detection of NB and treatment of refractory/recurrent NB. This is due to its specific targeting of surface noradrenaline transporters (NET-1), the expression of which is characteristic of neuroblasts in NB. 131I-mIBG shows promising responses in heavily pre-treated NB patients. Combinatorial approaches that enhance 131I-mIBG tumour uptake are of substantial clinical interest, but biomarkers of response are needed. The group investigated the potential of 18F-mFBG, a PET analogue of the 123I-mIBG radiotracer, to quantify NET-1 expression levels in mouse models of NB following treatment with AZD2014. This work supports the potential of 18F-mFBG to use this tracer in future studies for image-guided therapeutic strategies leading to more robust and durable responses to 131I-mIBG radiotherapy.6

Conclusion

The ongoing development of multimodal PET technology continues to drive preclinical oncology research in tracer development, therapy monitoring and tumour biology. The growing importance of immuno-oncology is augmented by the sophisticated PET imaging systems available on the market. Cutting-edge research is bringing the field one step closer to personalised treatment by using Affibody molecules as novel PET agents to target specific molecular pathways in tumour progression. Such stud­ies are vital to achieve the goal of a personalised medicine approach to optimise cancer treatment and patient care.

Figure 1. Radioconjugate uptake in xenografts with varying EGFR expression. Representative whole-body sagittal PET/CT images acquired 3 hours after injection. (Reproduced with permission from reference 5 in accordance with Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/.)
Figure 2. 18F-AlF-NOTA- ZEGFR:03115 uptake assessed 1 h after injection. Representative sagittal whole-body PET/CT images of mice bearing HN5 tumours (outlined on image) with or without treatment with cetuximab. (Reproduced with permission from reference 5 in accordance with Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/.)

References

  1. Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013 (https://www.ncbi.nlm.nih.gov/books/NBK5923/).
  2. Croteau E, Renaud JM, Richard M, Ruddy TD, Bénard F and deKemp RA (2016) PET Metabolic Biomarkers for Cancer, Biomarkers in Cancer, 8(S2): 61-69.
  3. Muller C, Schibli R (2011) Folic acid conjugates for nuclear imaging of folate receptor-positive cancer. J Nucl Med. 52:1-4.
  4. Schieferstein H, Ross TL (2014) A polar 18F-labeled amino acid derivative for click labeling of biomolecules. Eur. J. Org. Chem. 17: 3546-3550.
  5. Burley, T.A. Da Pieve, C. Martins, C.D. Ciobota, D.M. Allott, L. Oyen, W.J. Harrington, K.J. Smith, G. Kramer-Marek, G. (2019). Affibody-Based PET Imaging to Guide EGFR-Targeted Cancer Therapy in Head and Neck Squamous Cell Cancer Models. Journal of Nuclear Medicine, Vol.60 (3), pp. 353-361.
  6. Turnock, S, Turton DR, Martins CD, Chesler L, Wilson TC, Gouverneur V, Smith G, and & Kramer‑Marek G (2020) 18F‑meta‑fluorobenzylguanidine (18F‑mFBG) to monitor changes in norepinephrine transporter expression in response to therapeutic intervention in neuroblastoma models, Scientific Reports, 10:20918.

About the Author

Mette Lauritzen, PhD, joined Bruker BioSpin in 2020 as Market Product Manager for Nuclear Molecular Imaging. She has an academic background in preclinical MRI and PET Imaging, having completed post-doctoral positions at the Danish Research Center for Magnetic Resonance (DRCMR) and Stanford University, School of Medicine’s Radiology Department.

 

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