The low response rates associated with immune checkpoint inhibitor (ICI) use has led to a surge in research investigating adjuvant combination strategies in an attempt to enhance efficacy. ...Repurposing existing drugs as adjuvants accelerates the pace of cancer immune therapy research; however, many combinations exacerbate the immunogenic response elicited by ICIs and can lead to adverse immune-related events. Metformin, a widely used type 2 diabetes drug is an ideal candidate to repurpose as it has a good safety profile and studies suggest that metformin can modulate the tumour microenvironment, promoting a favourable environment for T cell activation but has no direct action on T cell activation on its own. In the current study we used PET imaging with 18FAlF-NOTA-KCNA3P, a radiopharmaceutical specifically targeting KV1.3 the potassium channel over-expressed on active effector memory T-cells, to determine whether combining PD1 with metformin leads to an enhanced immunological memory response in a preclinical colorectal cancer model. Flow cytometry was used to assess which immune cell populations infiltrate the tumours in response to the treatment combination. Imaging with 18FAlF-NOTA-KCNA3P demonstrated that adjuvant metformin significantly improved anti-PD1 efficacy and led to a robust anti-tumour immunological memory response in a syngeneic colon cancer model through changes in tumour infiltrating effector memory T-cells.
Protein-based
F-PET tracers offer new possibilities in early disease detection and personalized medicine. Their development relies heavily on the availability and effectiveness of
F-prosthetic ...groups. We prepared and evaluated a novel arginine-selective prosthetic group, 4-
Ffluorophenylglyoxal (
FFPG).
FFPG was radiosynthesized by a one-pot, two-step procedure with a non-decay-corrected (n.d.c.) isolated radiochemical yield (RCY) of 41 ± 8% (
= 10).
FFPG constitutes a generic tool for
F-labeling of various proteins, including human serum albumin (HSA), ubiquitin, interleukin-2, and interleukin-4 in ∼30-60% n.d.c. isolated RCYs.
FFPG conjugation with arginine residues is highly selective, even in the presence of a large excess of lysine, cysteine, and histidine.
FFPG protein conjugates are able to preserve the binding affinity of the native proteins while also demonstrating excellent
stability. The
FFPG-HSA conjugate has prolonged blood retention, which can be applied as a potential blood pool PET imaging agent. Thus,
FFPG is an arginine-selective bioconjugation reagent that can be effectively used for the development of
F-labeled protein radiopharmaceuticals.
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. 18FFDG is able to detect activated beige fat but provides little information on unstimulated beige fat mass. We have assessed the use of 18FFEPPA, 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.
Female Balb/c mice (n = 5) were treated for 7 days with the β3 adrenergic agonist CL-316,243 to induce the browning of inguinal WAT (beige fat). Animals were imaged longitudinally with 18FFDG and 18FFEPPA 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.
Repeated dosing with β3-adrenergic agonist CL-316,243 caused a significant increase in 18FFDG uptake in both interscapular BAT and inguinal WAT associated with the increased metabolic activity of brown and beige adipocytes respectively. 18FFEPPA 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 18FFEPPA uptake is associated with the expression of mitochondria in BAT and beige adipocytes and independent of activation.
These data show that 18FFEPPA 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.
•TSPO-18kDa tracers can detect BAT under non-stimulated, thermoneutral conditions.•TSPO-18kDa tracers can detect the formation of beige adipocytes in white adipose tissue.•TSPO-18kDa tracers may aid in the development of new approaches to treat obesity.
Immune checkpoint inhibitors have shown great promise, emerging as a new pillar of treatment for cancer; however, only a relatively small proportion of recipients show a durable response to ...treatment. Strategies that reliably differentiate durably-responding tumours from non-responsive tumours are a critical unmet need. Persistent and durable immunological responses are associated with the generation of memory T cells. Effector memory T cells associated with tumour response to immune therapies are characterized by substantial upregulation of the potassium channel Kv1.3 after repeated antigen stimulation. We have developed a new Kv1.3 targeting radiopharmaceutical,
FAlF-NOTA-KCNA3P, and evaluated whether it can reliably differentiate tumours successfully responding to immune checkpoint inhibitor (ICI) therapy targeting PD-1 alone or combined with CLTA4. In a syngeneic colon cancer model, we compared tumour retention of
FAlF-NOTA-KCNA3P with changes in the tumour immune microenvironment determined by flow cytometry. Imaging with
FAlF-NOTA-KCNA3P reliably differentiated tumours responding to ICI therapy from non-responding tumours and was associated with substantial tumour infiltration of T cells, especially Kv1.3-expressing CD8
effector memory T cells.
Hepatocellular carcinoma (HCC) is a notoriously difficult cancer to treat. The recent development of immune checkpoint inhibitors has revolutionised HCC therapy; however, successful response is only ...observed in a small percentage of patients. Biomarkers typically used to predict treatment response in other tumour types are ineffective in HCC, which arises in an immune-suppressive environment. However, imaging markers that measure changes in tumour infiltrating immune cells may supply information that can be used to determine which patients are responding to therapy posttreatment. We have evaluated 18FAlF-mNOTA-GZP, a radiolabeled peptide targeting granzyme B, to stratify response to ICIs in a HEPA 1-tumours, a syngeneic model of HCC. Posttherapy, in vivo tumour retention of 18FAlF-mNOTA-GZP was correlated to changes in tumour volume and tumour-infiltrating immune cells. 18FAlF-mNOTA-GZP successfully stratified response to immune checkpoint inhibition in the syngeneic HEPA 1-6 model. FACS indicated significant changes in the immune environment including a decrease in immune suppressive CD4+ T regulatory cells and increases in tumour-associated GZB+ NK+ cells, which correlated well with tumour radiopharmaceutical uptake. While the immune response to ICI therapies differs in HCC compared to many other cancers, 18FAlF-mNOTA-GZP retention is able to stratify response to ICI therapy associated with tumour infiltrating GZB+ NK+ cells in this complex tumour microenvironment.
Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for immune evasion, allowing immune cells to target and destroy cancer cells. Despite rapid advancements in ...immunotherapy, durable response rates to ICIs remains low. To address this, combination clinical trials are underway assessing whether adjuvants can enhance responsiveness by increasing tumour immunogenicity. CpG-oligodeoxynucleotides (CpG-ODN) are synthetic DNA fragments containing an unmethylated cysteine-guanosine motif that stimulate the innate and adaptive immune systems by engaging Toll-like receptor 9 (TLR9) present on the plasmacytoid dendritic cells (pDCs) and B cells. Here, we have assessed the ability of AlF-mNOTA-GZP, a peptide tracer targeting granzyme B, to serve as a PET imaging biomarker in response to CpG-ODN 1585 in situ vaccine therapy delivered intratumourally (IT) or intraperitoneally (IP) either as monotherapy or in combination with αPD1.
FAlF-mNOTA-GZP was able to differentiate treatment responders from non-responders based on tumour uptake. Furthermore,
FAlF-mNOTA-GZP showed positive associations with changes in tumour-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells, and decreases in suppressive F4/80+ cells.
FAlF-mNOTA-GZP tumour uptake was mediated by GZB expressing CD8+ cells and successfully stratifies therapy responders from non-responders, potentially acting as a non-invasive biomarker for ICIs and combination therapy evaluation in a clinical setting.
Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a severe complication of malaria that occurs despite effective antimalarial treatment. Currently, noninvasive imaging procedures ...such as chest X-rays are used to assess edema in established MA-ARDS, but earlier detection methods are needed to reduce morbidity and mortality. The early stages of MA-ARDS are characterized by the infiltration of leukocytes, in particular monocytes/macrophages; thus, monitoring of immune infiltrates may provide a useful indicator of early pathology. In this study, Plasmodium berghei ANKA-infected C57BL/6 mice, a rodent model of MA-ARDS, were longitudinally imaged using the 18-kDa translocator protein (TSPO) imaging agent
FFEPPA as a marker of macrophage accumulation during the development of pathology and in response to combined artesunate and chloroquine diphosphate (ART+CQ) therapy.
FFEPPA uptake was compared to blood parasitemia levels and to levels of pulmonary immune cell infiltrates by using flow cytometry. Infected animals showed rapid increases in lung retention of
FFEPPA, correlating well with increases in blood parasitemia and pulmonary accumulation of interstitial inflammatory macrophages and major histocompatibility complex class II (MHC-II)-positive alveolar macrophages. Treatment with ART+CQ abrogated this increase in parasitemia and significantly reduced both lung uptake of
FFEPPA and levels of macrophage infiltrates. We conclude that retention of
FFEPPA in the lungs is well correlated with changes in blood parasitemia and levels of lung-associated macrophages during disease progression and in response to ART+CQ therapy. With further development, TSPO biomarkers may have the potential to accurately assess the early onset of MA-ARDS.
Purpose
The aim of our study was to compare the incidence of idiopathic central precocious puberty (CPP) in our highly specialized Endocrinological Center before and after the onset of COVID-19 ...lockdown; we also aimed to identify any potential difference between girls with CPP from the two different time periods.
Methods
We retrospectively analyzed the auxological profile of 49 girls with idiopathic CPP: 30 with pre-lockdown onset and 19 with post-lockdown onset of the disease. We collected patients’ characteristics (medical history, physical examination, baseline and dynamic hormonal assessment, bone age, pelvic ultrasound) and compared them between the two groups.
Results
We registered an almost threefold increase in CPP incidence in the 2020–2021 period compared to the previous six years. In post-lockdown patients we found a trend for an earlier diagnosis in terms of both chronological age (
p
0.0866) and days between the onset of first pubertal signs and diagnosis (
p
0.0618). We also found that post-lockdown patients had a significantly lower hypothalamus-pituitary–gonadal axis activation (lower ∆LH% after GnRH test,
p
0.0497), a significantly lower increase in bone age calculated at RUS with TW3 method (
p
0.0438) and a significantly reduced ovarian activation in females (lower delta-4-androstenedione levels, p 0.0115). Interestingly, post-lockdown patients were born from mothers with an older age at menarche (
p
0.0039).
Conclusions
Besides confirming a significant increase in new diagnoses of CPP in the post-lockdown period, our findings among Post-lockdown girls also suggest a less progressive form of CPP and a stronger environmental influence compared to genetic background in determining the timing of pubertal onset.
Purpose
Chemotherapeutic adjuvants, such as oxaliplatin (OXA) and 5-fluorouracil (5-FU), that enhance the immune system, are being assessed as strategies to improve durable response rates when used ...in combination with immune checkpoint inhibitor (ICI) monotherapy in cancer patients. In this study, we explored granzyme B (GZB), released by tumor-associated immune cells, as a PET imaging-based stratification marker for successful combination therapy using a fluorine-18 (
18
F)-labelled GZB peptide (
18
FAlF-mNOTA-GZP).
Methods
Using the immunocompetent CT26 syngeneic mouse model of colon cancer, we assessed the potential for
18
FAlF-mNOTA-GZP to stratify OXA/5-FU and ICI combination therapy response
via
GZB PET.
In vivo
tumor uptake of
18
FAlF-mNOTA-GZP in different treatment arms was quantified by PET, and linked to differences in tumor-associated immune cell populations defined by using multicolour flow cytometry.
Results
18
FAlF-mNOTA-GZP tumor uptake was able to clearly differentiate treatment responders from non-responders when stratified based on changes in tumor volume. Furthermore,
18
FAlF-mNOTA-GZP showed positive associations with changes in tumor-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells and GZB+ NK+ cells.
Conclusions
18
FAlF-mNOTA-GZP tumor uptake, driven by changes in immune cell populations expressing GZB, is able to stratify tumor response to chemotherapeutics combined with ICIs. Our results show that, while the immunomodulatory mode of action of the chemotherapies may be different, the ultimate mechanism of tumor lysis through release of Granzyme B is an accurate biomarker for treatment response.
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