Institute of Molecular and Translational Medicine

PurposeComplete and minimally invasive cancer surgery remains challenging. Targeting the fibroblast activation protein (FAP) offers valuable opportunities for surgical planning, intraoperative guidance and improved resection outcomes. Herein, we developed the first dimeric, dual-modality FAP-targeted imaging agents and investigated the influence of different near-infrared cyanine-7 dyes on their final properties.MethodsFour dual-modality ligands based on the Fusarinine C scaffold were synthesized. Their FAP specificity and retention were evaluated in cellular and xenograft tumor models. The most promising candidates were labelled with 67/68Ga and assessed in vivo at early time points by PET/CT imaging and by comparative SPECT/CT and NIR fluorescence imaging (FI) up to two days post-injection.ResultsDistinct fluorophore influences on the properties of the final compounds were identified. The introduction of the s775z dye demonstrated a beneficial effect on the cellular uptake and on the in vivo biodistribution profile as revealed by the greatest improvement in blood clearance and the least off-target accumulation in liver and kidneys when compared to the control and to the other candidates respectively. Ex vivo experiments and in vivo PET/CT, SPECT/CT and FI studies in xenografted mice confirmed these findings and demonstrated sustained tumor uptake (> 7% ID/g and > 5% ID/g at 1 h and 1 day p.i. respectively) for 67Ga-s775z-FFAPi and 67Ga-IRDye-FFAPi.ConclusionsIn this study we introduced and evaluated novel dimeric FAP-targeting agents for dual-modality applications. In the preclinical setting, within the group of compounds investigated, two candidates enabled tumor visualization through PET, SPECT and optical imaging, providing satisfactory background contrast after a single administration and supporting their potential for preoperative nuclear imaging and subsequent fluorescence-guided surgery.

Tau aggregation driven by microtubule‐associated protein tau (MAPT) mutations is central to frontotemporal dementia pathology, yet no disease‐modifying therapies effectively target mutant tau. Here, we identify purpurin (PUR) and oleocanthal (OLC) as selective inhibitors of mutant tau aggregation using peptide models spanning the R2R3 interface. Biophysical and cellular assays demonstrated that both compounds more effectively inhibit the aggregation of mutant tau peptides compared to wild‐type, with PUR preferentially targeting V287I and N279K variants, and OLC showing broader inhibitory activity. Surface plasmon resonance and docking analyses revealed more stable interactions and lower binding free energies with mutant tau, consistent with their enhanced inhibitory effects. Computational studies using monomeric and fibrillar tau structures supported the mutation‐specific binding profiles of PUR and OLC. Atomic force microscopy and confocal imaging confirmed reduced fibril formation, while post‐transduction treatment assays showed that both compounds significantly suppressed intracellular tau propagation. Additionally, OLC reduced tau phosphorylation and oligomerization in SY5Y‐TauP301L‐EGFP cells expressing mutant tau. These findings highlight the potential of PUR and OLC as structurally distinct, mutation‐targeted inhibitors of tau aggregation and propagation, providing a rationale for their further development as candidate therapeutics for frontotemporal dementia.

The evolutionarily important Hedgehog (HH) signaling pathway plays a critical role in the development and progression of multiple solid tumors, such as basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, and various gastrointestinal, pulmonary, and brain tumors. The proteins of the Gli (glioma-associated oncogene homologue) family are key mediators of the HH pathway. In the present study, we have focused on triterpenoid derivatives, which have been shown to induce apoptosis and inhibit HH signaling in rhabdomyosarcoma. Utilizing a U-87MG glioblastoma-derived reporter cell line, we screened a structurally diverse library of triterpenoid derivatives to identify potential antagonists of Gli-mediated transcription. We revealed two derivatives that not only selectively inhibited Gli-mediated gene transactivation but also displayed greater potency than the known Gli1 inhibitor GANT61. These compounds also demonstrated dose- and time-dependent inhibition of U-87MG tumor cell proliferation in vitro. Further mechanistic studies provided genetic evidence for the inhibition of the downstream HH pathway by these compounds, via reduced expression of Gli1 and its transcription targets. However, these compounds did not affect the ciliary localization of Smoothened (Smo). Our findings suggest that the observed inhibitory effects are likely due to a direct interaction between our compounds and Gli1.

To assess the potential of αvβ6-integrin as a theranostic target in esophageal cancer. Membranous β6-integrin (ITGB6) expression was analyzed in 306 specimens of human esophageal squamous cell carcinoma (ESCC) obtained by immunohistochemistry (IHC) from 100 patient cases (1, 37, 58, and 4 of grade G1, G2, G3, and G4, respectively). Ga-68 labeling of D0103 was done manually for preclinical experiments and fully automated for clinical application. Preclinical characterization of Ga-68-D0103 was performed in SCID mice bearing subcutaneous xenografts of H2009 (αvβ6-positive) or MDA-MB-231 (αvβ6-negative) carcinoma cell lines, by ex vivo biodistribution (10, 30, 90, and 180min p.i) and PET imaging (30, 90, and 180min p.i.)., without and with co-injection of gelofusine (4% succinylated gelatin). A patient with type-II diabetes (f, 68y, 115kg) with proximal G2 ESCC was investigated by Ga-68-D0103 PET/CT (193MBq) at 15, 45, 90, and 104min p.i.. 99% of ESCC cases were found β6-integrin positive by IHC, of which 48%, 31%, and 20% showed strong, moderate, and low ITGB6 expression, respectively, with no correlation to tumor grade. Ex vivo biodistribution of Ga-68-D0103 in H2009 xenografted mice after 30, 90, and 180min showed tumor-to-blood ratios of 6.8, 37, and 124, respectively; tumor-to-muscle ratios of 12, 14, and 36, respectively; tumor-to-liver ratios of 10, 17, and 14, respectively; and tumor-to-pancreas ratios of 20, 47, and 56, respectively. Co-administration of gelofusine did not change the tumor uptake but reduced the kidney uptake by 89% (from 178%iA/g to 19.1%iA/g, 90min p.i.), resulting in an 8.7-fold higher tumor/kidney ratio. µPET imaging in H2009 xenografted mice confirmed a high tumor uptake and low background already 30min p.i.. Blockade biodistribution and µPET in αvβ6-(-) MDA-MB-231 mice demonstrated target specificity. Clinical PET/CT of a patient with ESCC showed increasing tracer uptake over time in the primary tumor (SUVmax 9.0 and 11.3 at 15 and 104min p.i., respectively) and in a lymph node metastasis (SUVmax 19.5 and 28.3, respectively), and a decreasing blood pool activity (SUVmean 2.75 and 0.98, respectively). High (99%) membranous expression frequency and density on tumor cells underscores the potential of αvβ6-integrin as a theranostic target in ESCC, suggesting that αvβ6-integrin PET/CT imaging may adopt a role in re-staging and therapy guidance in this cancer type. The prolonged tumor retention furthermore indicates a therapeutic potential of αvβ6-integrin targeted radiopharmaceuticals when labeled with radionuclides such as lutetium-177, terbium-161, or actinium-225.

The natural product colchicine (Col) is a medication used to treat severe inflammatory conditions. Although its mechanism of action at the level of the cytoskeleton is known, its subcellular distribution has not yet been properly studied. In this work, we present the first rational approach to assess the intracellular localization and biological activity of this alkaloid. We conjugated Col to green-emitting BODIPY dyes (CBs) with alternative linkers of different lengths (CB1-CB12) via different types of linkages. Connections of Col with BODIPY generally reduced its cytotoxicity to different levels depending on the type of linker. From the analysis of CB effects on cytotoxicity, cell cycle, and tubulin polymerization, we selected the most potent substances for fluorescence microscopy. Treatment of cells with 10 μM conjugates for 15 h showed different effects on microtubule organization. Live-cell imaging revealed that CBs rapidly associated with cellular membranes. Double label experiments unveiled that the CB4, which was the most effective in inhibiting tubulin polymerization, binds to the endoplasmic reticulum (ER) and mitochondria. In silico modeling and SPR analyses confirmed the high potency of CB4 to bind to the colchicine site on tubulin.

The tumor suppressor gene NKX3.1 and the LPL gene are located in close proximity on chromosome 8, and their deletion has been reported in multiple studies. However, the significance of LPL loss may be misinterpreted due to its co-deletion with NKX3.1, a well-established event in prostate carcinogenesis. This study investigates whether LPL deletion represents a biologically relevant event or occurs merely as a bystander to NKX3.1 loss. We analyzed 28 formalin-fixed paraffin-embedded prostate cancer samples with confirmed LPL deletion and 28 without. Immunohistochemical staining was performed, and previously published whole-genome sequencing data from 103 prostate cancer patients were reanalyzed. Deletion of the 8p21.3 region was associated with higher Gleason grade groups. While NKX3.1 expression was significantly reduced in prostate cancer compared to benign prostatic hyperplasia, LPL protein expression showed no significant difference between cancerous and benign tissue, nor was it affected by the 8p21.3 deletion status. Copy number analysis confirmed the co-deletion of NKX3.1 and LPL in 54 patients. Notably, NKX3.1 loss without accompanying LPL deletion was observed in eight additional cases. These findings suggest that LPL deletion is a passenger event secondary to NKX3.1 loss and underscore the importance of cautious interpretation of cytogenetic findings involving the LPL locus.

CDD plays a pivotal role within the pyrimidine salvage pathway. In this study, a novel, rapid method for the identification of cell lines lacking functional cytidine deaminase was developed. This innovative method utilizes immunocytochemical detection of the product of 5-fluorocytidine deamination, 5-fluorouridine in cellular RNA, enabling the identification of these cells within two hours. The approach employs an anti-bromodeoxyuridine antibody that also specifically binds to 5-fluorouridine and its subsequent detection by a fluorescently labeled antibody. Our results also revealed a strong correlation between the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio and cytidine deaminase content. On the other hand, no correlation was observed between the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio and deoxycytidine monophosphate deaminase content. Similarly, no correlation was observed between this ratio and equilibrative nucleoside transporters 1 or 2. Finally, concentrative nucleoside transporters 1, 2, or 3 also do not correlate with the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio.

Vaccines represent an essential tool for the prevention of infectious diseases. Upon administration, a complex interaction occurs between the vaccine formulation and the recipient's immune system, ultimately resulting in protection against disease. Significant variability exists in individual and population responses to vaccination, and these differences remain the focus of the ongoing research. Notably, well-documented factors, such as age, gender, and genetic predisposition, influence immune responses. In contrast, the effects of overweight and obesity have not been as thoroughly investigated. The evidence indicates that a high body mass index (BMI) constitutes a significant risk factor for infections in general, with adipose tissue playing a crucial role in modulating the immune response. Furthermore, suboptimal levels of vaccine seroconversion have been observed among individuals with obesity. This review provides a plausible examination of the immunity and protection conferred by various vaccines in individuals with an overweight status, offering a comprehensive analysis of the mechanisms to enhance vaccination efficiency.