Purpose: Great advances have recently been made in treating patients with metastatic melanoma. However, existing therapies are less effective on cerebral than extracerebral metastases. This highlights the potential role of the brain environment on tumor progression and drug resistance and underlines the need for “brain-specific” therapies. We previously showed that the PI3K-AKT survival pathway is hyperactivated in brain but not extracerebral melanoma metastases and that astrocyte-conditioned medium activates AKT in melanoma cells in vitro. We therefore tested the PI3K inhibitor buparlisib as an antitumor agent for melanoma brain metastases. Experimental Design and Results: Buparlisib inhibited AKT activity, decreased proliferation, and induced apoptosis in metastatic melanoma cell lines and short-term brain melanoma cells, irrespective of their BRAF and NRAS mutation status. In addition, buparlisib inhibited hyperactivated AKT and induced apoptosis in melanoma cells that were stimulated with astrocyte-conditioned medium. The growth of tumors induced by injecting human BRAF- and NRAS-mutant metastatic melanoma cells into the brain of mice was significantly inhibited by buparlisib. Conclusions: These results emphasize the value of targeting the PI3K pathway as a strategy to develop drugs for melanoma brain metastases. Clin Cancer Res; 22(23); 5818–28. ©2016 AACR.

Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. Methods: We developed protocols that minimize the inhibitory effects of ⁶⁴Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (⁶⁴Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-γ–producing CD4⁺ T (Th1) cells with 0.7–2.2 MBq of ⁶⁴Cu-PTSM and analyzed cell viability, IFN-γ production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular ⁶⁴Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 10^{7 64}Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of ⁶⁴Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 10^{7 64}Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. Results: PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, ⁶⁴Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered ⁶⁴Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of ⁶⁴Cu-OVA-Th1 cells in the pulmonary LNs (6.8 ± 1.1 percentage injected dose per cubic centimeter [%ID/cm³]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity–diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 ± 0.4 %ID/cm³). As expected, ⁶⁴Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 ± 0.5 %ID/cm³) when compared with phosphate-buffered saline–challenged animals (4.6 ± 0.5 %ID/cm³). Conclusion: Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for ⁶⁴Cu-PTSM–labeled T cells in clinical trials and novel therapy concepts focusing on T-cell–based immunotherapies of autoimmune diseases or cancer.

Differential diagnosis and therapy of heterogeneous breast tumors poses a major clinical challenge. To address the need for a comprehensive, noninvasive strategy to define the molecular and functional profiles of tumors in vivo, we investigated a novel combination of metabolic PET and diffusion-weighted (DW)-MRI in the polyoma virus middle T antigen transgenic mouse model of breast cancer. The implementation of a voxelwise analysis for the clustering of intra- and intertumoral heterogeneity in this model resulted in a multiparametric profile based on [18F]Fluorodeoxyglucose ([18F]FDG)-PET and DW-MRI, which identified three distinct tumor phenotypes in vivo, including solid acinar, and solid nodular malignancies as well as cystic hyperplasia. To evaluate the feasibility of this approach for clinical use, we examined estrogen receptor-positive and progesterone receptor-positive breast tumors from five patient cases using DW-MRI and [18F]FDG-PET in a simultaneous PET/MRI system. The postsurgical in vivo PET/MRI data were correlated to whole-slide histology using the latter traditional diagnostic standard to define phenotype. By this approach, we showed how molecular, structural (microscopic, anatomic), and functional information could be simultaneously obtained noninvasively to identify precancerous and malignant subtypes within heterogeneous tumors. Combined with an automatized analysis, our results suggest that multiparametric molecular and functional imaging may be capable of providing comprehensive tumor profiling for noninvasive cancer diagnostics. Cancer Res; 76(18); 5512–22. ©2016 AACR.

Quantification accuracy and partial volume effect (PVE) of the Siemens Inveon PET scanner were evaluated. The influence of transmission source activities (40 and 160 MBq) on the quantification accuracy and the PVE were determined. Dynamic range, object size and PVE for different sphere sizes, contrast ratios and positions in the field of view (FOV) were evaluated. The acquired data were reconstructed using different algorithms and correction methods. The activity level of the transmission source and the total emission activity in the FOV strongly influenced the attenuation maps. Reconstruction algorithms, correction methods, object size and location within the FOV had a strong influence on the PVE in all configurations. All evaluated parameters potentially influence the quantification accuracy. Hence, all protocols should be kept constant during a study to allow a comparison between different scans.

The increasing use of genetically engineered mice as animal models of human disease in biomedical research, latest advances in imaging technologies, and development of novel, highly specific radiolabeled biomarkers provide great potential to study receptor expression and gene function in vivo in mice. ¹¹C-raclopride is a widely used PET tracer to measure striatal D₂ receptor binding and was used to test the feasibility of the multiple-ligand-concentration receptor assay for D₂ receptor quantification. Methods: Mice underwent a total of 4 scans with decreasing specific activities from 141 to 0.4 GBq/μmol, corresponding to ¹¹C-raclopride injected doses of 2.4 to 1,274 nmol/kg, using either a standard bolus injection protocol (n = 12) or a bolus–plus–constant infusion protocol to attain true equilibrium conditions (n = 7). Receptor occupancy was plotted as a function of raclopride dose, and D₂ receptor density and raclopride affinity were calculated using linear and nonlinear regression analysis, respectively. In addition, we used ex vivo autoradiography, a more spatially accurate imaging technology, to validate the in vivo PET measurements, and we performed test–retest experiments to determine the reproducibility and reliability of the PET-derived measures. Results: The receptor occupancy curves showed that an injected tracer dose of 4.5 nmol/kg induces approximately 10% receptor occupancy, whereas 1% receptor occupancy will be achieved at tracer doses of approximately 0.45 nmol/kg. Using the bolus injection protocol and nonlinear regression analysis, we determined that the average D₂ receptor density was 9.6 ± 1.1 pmol/mL, and the apparent raclopride affinity was 5.0 ± 0.6 pmol/mL. These values agreed well with those obtained at true equilibrium conditions. In contrast, linear Scatchard analysis did not lead to the expected linear relationship because nonsaturable binding was observed at high raclopride concentrations, and thus, it seems to be unsuitable for quantitative ¹¹C-raclopride analysis in mice. Conclusion: Our data showed that the tracer mass, if higher than 4 nmol/kg, can strongly affect binding parameter estimations and must be considered when performing kinetic analysis, specifically in mice. We also demonstrated that the in vivo determination of D₂ receptor density and raclopride affinity is feasible in mice using multiple-injection protocols and nonlinear regression analysis.