Granzyme B PET Imaging of Immune Checkpoint Inhibitor Combinations in Colon Cancer Phenotypes.

PURPOSE: Immune checkpoint inhibitor (ICI) monotherapy and combination regimens are being actively pursued as strategies to improve durable response rates in cancer patients. However, the biology surrounding combination therapies is not well understood and may increase the likelihood of immune-mediated adverse events. Accurate stratification of ICI response by non-invasive PET imaging may help ensure safe therapy management across a wide number of cancer phenotypes. PROCEDURES: We have assessed the ability of a fluorine-labelled peptide, [18F]AlF-mNOTA-GZP, targeting granzyme B, to stratify ICI response in two syngeneic models of colon cancer, CT26 and MC38. In vivo tumour uptake of [18F]AlF-mNOTA-GZP following ICI monotherapy, or in combination with PD-1 was characterised and correlated with changes in tumour-associated immune cell populations. RESULTS: [18F]AlF-mNOTA-GZP showed good predictive ability and correlated well with changes in tumour-associated T cells, especially CD8+ T cells; however, overall uptake and response to monotherapy or combination therapies was very different in the CT26 and MC38 tumours, likely due to the immunostimulatory environment imbued by the MSI-high phenotype in MC38 tumours. CONCLUSIONS: [18F]AlF-mNOTA-GZP uptake correlates well with changes in CD8+ T cell populations and is able to stratify tumour response to a range of ICIs administered as monotherapies or in combination. However, tracer uptake can be significantly affected by preexisting phenotypic abnormalities potentially confusing data interpretation.

Imaging Fibrogenesis in a Diet-Induced Model of Nonalcoholic Steatohepatitis (NASH).

Purpose: Liver fibrosis is the hallmark of chronic nonalcoholic steatohepatitis (NASH) and is characterised by the excessive deposition of extracellular matrix proteins. Early detection and accurate staging of liver fibrosis is critically important for patient management. One of the earliest pathological markers in NASH is the activation of hepatic stellate cells (HSCs) which may be exploited as a marker of fibrogenesis. Activated HSCs secreting factors such as integrin α v ß 3 propagate fibrosis. The purpose of the current study was to assess the utility of the integrin α v ß 3 imaging agent [18F]FtRGD for the early detection of fibrosis in a diet-induced model of NASH longitudinally using PET imaging. Procedures: Mice were fed with either standard chow diet (SD), high-fat diet (HFD), or a choline-deficient, L-amino acid-defined high-fat fibrogenic diet (CDAHFD) to mimic the clinical pathology of liver disease and followed longitudinally for 10 weeks to assess the development of liver fibrosis using [18F]FtRGD positron emission tomography (PET) imaging. Standard blood biochemistry, histological measures, and qPCR were used to quantify integrin α v ß 3, smooth muscle actin, and collagen types 1 and 6 to assess the extent of NASH pathology and accurately stage liver fibrosis. Results: The CDAHFD fibrogenic diet predictably developed hepatic inflammation and steatosis over the 10 weeks studied with little NASH pathology detected in high fat diet-treated animals. Stage 1 fibrosis was detected early by histology at day 21 and progressed to stage 2 by day 35 and stage 3 by day 56 in mice fed with CDAHFD diet only. Noninvasive imaging with [18F]FtRGD correlated well with integrin α v ß 3 and was able to distinguish early mild stage 2 fibrosis in CDAHFD animals compared with standard chow diet-fed animals at day 35. When compared with high fat diet-fed animals, [18F]FtRGD was only able to distinguish later moderate stage 2 fibrosis in CDAHFD animals at day 49. Conclusions: The diet-induced progression of liver fibrosis was confirmed using histology and correlated well with the mRNA of integrin α v ß 3 and extracellular matrix protein expression. [18F]FtRGD showed very good correlation between liver uptake and integrin α v ß 3 expression and similar detection sensitivity to the current clinical gold standard modalities for staging of liver fibrosis.

Imaging adipose tissue browning using the TSPO-18kDa tracer [18F]FEPPA.

OBJECTIVES: The browning of white adipose tissue (WAT) into beige has been proposed as a strategy to enhance energy expenditure to combat the growing epidemic of obesity. Research into browning strategies are hampered by the lack of sensitive, translatable, imaging tools capable of detecting beige fat mass non-invasively. [18F]FDG is able to detect activated beige fat but provides little information on unstimulated beige fat mass. We have assessed the use of [18F]FEPPA, a tracer for the TSPO-18KDa found on the outer mitochondrial membrane, as an alternative imaging agent capable of detecting unstimulated brown fat (BAT) and beige fat. METHODS: Female Balb/c mice (n = 5) were treated for 7 days with the ß3 adrenergic agonist CL-316,243 to induce the browning of inguinal WAT (beige fat). Animals were imaged longitudinally with [18F]FDG and [18F]FEPPA and uptake in interscapular BAT and inguinal WAT assessed. The browning of inguinal WAT was confirmed using H&E and immunohistochemical detection of UCP-1 and TSPO. RESULTS: Repeated dosing with ß3-adrenergic agonist CL-316,243 caused a significant increase in [18F]FDG uptake in both interscapular BAT and inguinal WAT associated with the increased metabolic activity of brown and beige adipocytes respectively. [18F]FEPPA uptake was likewise increased in inguinal WAT but showed no increase in BAT uptake due to stimulation over the same time course. Furthermore, inguinal WAT uptake was unaffected by pharmacological blockade, indicating that [18F]FEPPA uptake is associated with the expression of mitochondria in BAT and beige adipocytes and independent of activation. CONCLUSION: These data show that [18F]FEPPA can detect BAT and newly formed beige fat under non-stimulated, thermoneutral conditions and that uptake after stimulation is linked to mitochondrial expression as opposed to activation.

Imaging the Proangiogenic Effects of Cardiovascular Drugs in a Diabetic Model of Limb Ischemia.

Purpose: Peripheral artery disease (PAD) causes narrowing of arteries in the limbs, leading to tissue ischemia, gangrene, and eventually limb amputation. The presence of diabetes greatly exacerbates the course of PAD, accounting for the majority of lower limb amputations. Therapeutic strategies focussing on macrovascular repair are less effective in diabetic patients where smaller vessels are affected, and proangiogenic therapies offer a viable adjunct to improve vascularisation in these at risk individuals. The purpose of the current study was to assess the proangiogenic effects of drugs routinely used to treat cardiovascular disease in a diabetic murine model of hind limb ischemia longitudinally using multimodal imaging. Procedures: Diabetic mice underwent surgical intervention to induce hind limb ischemia and were treated with simvastatin, metformin, or a combination orally for 28 days and compared to diabetic and nondiabetic mice. Neovascularisation was assessed using [18F]FtRGD PET imaging, and macrovascular volume was assessed by quantitative time of flight MRI. At each imaging time point, VEGF expression and capillary vessel density were quantified using immunohistochemical analysis, and functional recovery and disease progression were assessed. Results: Combined use of simvastatin and metformin significantly increased neovascularisation above levels measured with either treatment alone. Early angiogenic events were accurately assessed using PET [18F]FtRGD, showing maximal retention in the ischemic hind limb by day 8, which translated to a sustained increase in vascular volume at later time points. Immunohistochemical analysis shows that combined therapy significantly increased VEGF expression and capillary density (CD31+) in a similar time course and also slowed disease progression while simultaneously improving functional foot use. Conclusions: Combined treatment with simvastatin and metformin led to a significant improvement in limb angiogenesis, vascular volume, and sustained functional recovery in a diabetic murine model of HLI. PET imaging with [18F]FtRGD provides a robust method for early detection of these proangiogenic effects preclinically and may be useful for the assessment of proangiogenic therapies used clinically to treat diabetic PAD patients.