Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. Increased expression of CXCR4 has been associated with liver metastasis, disease progression, and shortened survival. Using in vitro cell binding studies and the in ovo model, we aimed to investigate the potential of [⁶⁸Ga]Ga-Pentixafor, a radiotracer specifically targeting human CXCR4, for CRC imaging. Specific membrane binding and internalisation of [⁶⁸Ga]Ga-Pentixafor was shown for HT29 cells, but not for HCT116 cells. Accordingly, [⁶⁸Ga]Ga-Pentixafor accumulated specifically in CAM-xenografts derived from HT29 cells, but not in HCT116 xenografts, as determined by µPET/MRI. The CAM-grown xenografts were histologically characterised, demonstrating vascularisation of the graft, preserved expression of human CXCR4, and viability of the tumour cells within the grafts. In vivo viability was further confirmed by µPET/MRI measurements using 2-[¹⁸F]FDG as a surrogate for glucose metabolism. [⁶⁸Ga]Ga-Pentixafor µPET/MRI scans showed distinct radiotracer accumulation in the chick embryonal heart, liver, and kidneys, whereas 2-[¹⁸F]FDG uptake was predominantly found in the kidneys and joints of the chick embryos. Our findings suggest that [⁶⁸Ga]Ga-Pentixafor is an interesting novel radiotracer for CRC imaging that is worth further investigation. Moreover, this study further supports the suitability of the CAM-xenograft model for the initial preclinical evaluation of targeted radiopharmaceuticals.

Combining therapeutic with diagnostic agents (theranostics) can revolutionize the course of malignant diseases. Chemotherapy, hyperthermia, or radiation are used together with diagnostic methods such as magnetic resonance imaging (MRI). In contrast to conventional contrast agents (CAs), which only enable non-specific visualization of tissues and organs, the theranostic probe offers targeted diagnostic imaging and therapy simultaneously. Methods: Novel salinomycin (Sal)-based theranostic probes comprising two different paramagnetic metal ions, gadolinium(III) (Gd(III)) or manganese(II) (Mn(II)), as signal emitting motifs for MRI were synthesized and characterized by elemental analysis, infrared spectral analysis (IR), electroparamagnetic resonance (EPR), thermogravimetry (TG) differential scanning calorimetry (DSC) and electrospray ionization mass spectrometry (ESI-MS). To overcome the water insolubility of the two Sal-complexes, they were loaded into empty bacterial ghosts (BGs) cells as transport devices. The potential of the free and BGs-loaded metal complexes as theranostics was evaluated by in vitro relaxivity measurements in a high-field MR scanner and in cell culture studies. Results: Both the free Sal-complexes (Gd(III) salinomycinate (Sal-Gd(III) and Mn(II) salinomycinate (Sal-Mn(II)) and loaded into BGs demonstrated enhanced cytotoxic efficacy against three human tumor cell lines (A549, SW480, CH1/PA-1) relative to the free salinomycinic acid (Sal-H) and its sodium complex (Sal-Na) applied as controls with IC₅₀ in a submicromolar concentration range. Moreover, Sal-H, Sal-Gd(III), and Sal-Mn(II) were able to induce perturbations in the cell cycle of treated colorectal and breast human cancer cell lines (SW480 and MCF-7, respectively). The relaxivity (r₁) values of both complexes as well as of the loaded BGs, were higher or comparable to the relaxivity values of the clinically applied contrast agents gadopentetate dimeglumine and gadoteridol. Conclusion: This research is the first assessment that demonstrates the potential of Gd(III) and Mn(II) complexes of Sal as theranostic agents for MRI. Due to the remarkable selectivity and mode of action of Sal as part of the compounds, they could revolutionize cancer therapy and allow for early diagnosis and monitoring of therapeutic follow-up.

Tumor neoangiogenesis is an important hallmark of cancer progression, triggered by alternating selective pressures from the hypoxic tumor microenvironment. Non-invasive, non-contrast-enhanced multiparametric MRI combining blood-oxygen-level-dependent (BOLD) MRI, which depicts blood oxygen saturation, and intravoxel-incoherent-motion (IVIM) MRI, which captures intravascular and extravascular diffusion, can provide insights into tumor oxygenation and neovascularization simultaneously. Our objective was to identify imaging markers that can predict hypoxia-induced angiogenesis and to validate our findings using multiplexed immunohistochemical analyses. We present an in vivo study involving 36 female athymic nude mice inoculated with luminal A, Her2+, and triple-negative breast cancer cells. We used a high-field 9.4-tesla MRI system for imaging and subsequently analyzed the tumors using multiplex immunohistochemistry for CD-31, PDGFR-β, and Hif1-α. We found that the hyperoxic-BOLD-MRI-derived parameter ΔR2* discriminated luminal A from Her2+ and triple-negative breast cancers, while the IVIM-derived parameter fIVIM discriminated luminal A and Her2+ from triple-negative breast cancers. A comprehensive analysis using principal-component analysis of both multiparametric MRI- and mpIHC-derived data highlighted the differences between triple-negative and luminal A breast cancers. We conclude that multiparametric MRI combining hyperoxic BOLD MRI and IVIM MRI, without the need for contrast agents, offers promising non-invasive markers for evaluating hypoxia-induced angiogenesis.

“Hot spot” ¹⁹F magnetic resonance imaging (MRI) has garnered significant attention recently for its ability to image various disease markers quantitatively. Unlike conventional gadolinium-based MRI contrast agents, which rely on proton signal modulation, ¹⁹F-MRI’s direct detection has a unique advantage in vivo, as the human body exhibits a negligible background ¹⁹F-signal. However, existing perfluorocarbon (PFC) or PFC-based contrast materials suffer from several limitations, including low longitudinal relaxation rates and relatively low imaging efficiency. Hence, we designed a macromolecular contrast agent featuring a high number of magnetically equivalent ¹⁹F-nuclei in a single macromolecule, adequate fluorine nucleus mobility, and excellent water solubility. This design utilizes superfluorinated polyphosphazene (PPz) polymers as the ¹⁹F-source; these are modified with sodium mercaptoethanesulfonate (MESNa) to achieve water solubility exceeding 360 mg/mL, which is a similar solubility to that of sodium chloride. We observed substantial signal enhancement in MRI with these novel macromolecular carriers compared to non-enhanced surroundings and aqueous trifluoroacetic acid (TFA) used as a positive control. In conclusion, these novel water-soluble macromolecular carriers represent a promising platform for future MRI contrast agents.

Hyperoxic BOLD-MRI targeting tumor hypoxia may provide imaging biomarkers that represent breast cancer molecular subtypes without the use of injected contrast agents. However, the diagnostic performance of hyperoxic BOLD-MRI using different levels of oxygen remains unclear. We hypothesized that molecular subtype characterization with hyperoxic BOLD-MRI is feasible independently of the amount of oxygen. Twenty-three nude mice that were inoculated into the flank with luminal A (n = 9), Her2⁺ (n = 5), and triple-negative (n = 9) human breast cancer cells were imaged using a 9.4 T Bruker BioSpin system. During BOLD-MRI, anesthesia was supplemented with four different levels of oxygen (normoxic: 21%; hyperoxic: 41%, 71%, 100%). The change in the spin–spin relaxation rate in relation to the normoxic state, $\Delta R_2{}^{*}$, dependent on the amount of erythrocyte-bound oxygen, was calculated using in-house MATLAB code. $\Delta R_2{}^{*}$ was significantly different between luminal A and Her2⁺ as well as between luminal A and triple-negative breast cancer, reflective of the less aggressive luminal A breast cancer’s ability to better deliver oxygen-rich hemoglobin to its tissue. Differences in $\Delta R_2{}^{*}$ between subtypes were independent of the amount of oxygen, with robust distinction already achieved with 41% oxygen. In conclusion, hyperoxic BOLD-MRI may be used as a biomarker for luminal A breast cancer identification without the use of exogenous contrast agents.

This study evaluates the performance of a positron emission tomography (PET) insert combined with a 9.4 Tesla magnetic resonance (MR) imaging system using three different radiofrequency (RF) coils. The impact of the RF coils within the PET field of view (FoV) on key PET performance parameters was carefully examined. Additionally, the accuracy of MR-based attenuation correction (AC) on the quantitative PET results was assessed. The performance of the PET scanner was assessed according to the NEMA NU 4-2008 standards, focusing on sensitivity, spatial resolution, noise-equivalent count (NEC) rate, and image quality metrics (homogeneity, spillover ratios, and recovery coefficients). These evaluations were conducted with three different RF coils within the PET FoV: a mouse-dedicated coil, a rat-dedicated coil (not optimized for PET), and a big-rat-dedicated coil. Quantitative testing of AC accuracy was also performed using homogeneously filled phantoms of varying sizes and activity levels, comparing images reconstructed with and without AC. The peak absolute sensitivity varied depending on the RF coil used, though spatial resolution was unaffected by the presence of the coils. The NEC curve for the mouse phantom peaked at 441.2 kcps at 29.3 MBq with the mouse-dedicated coil. For the rat phantom, NEC was only tested with the rat-dedicated coils, and the NEC peak reached 203.1 kcps at 27.5 MBq with the big-rat-dedicated coil. Activity concentrations in the NEMA image quality phantom were underestimated when standard MR-AC was applied, likely due to the absence of phantom walls in the generated µ-maps. However, for uniformly filled phantoms with wall thicknesses under 1 mm, standard MR-AC provided accurate quantification. Overall, the presence of RF coils had only a minor effect on the performance of the PET/MR insert, with the most significant variation observed for the non-PET-optimized coil. Attenuation correction based on generated µ-maps performed well for phantoms with minimal wall thickness.