Quantitative 18F-AV1451 Brain Tau PET Imaging in Cognitively Normal Older Adults, Mild Cognitive Impairment, and Alzheimer's Disease Patients

Recent developments of tau Positron Emission Tomography (PET) allows assessment of regional neurofibrillary tangles (NFTs) deposition in human brain. Among the tau PET molecular probes, 18F-AV1451 is characterized by high selectivity for pathologic tau aggregates over amyloid plaques, limited non-specific binding in white and gray matter, and confined off-target binding. The objectives of the study are (1) to quantitatively characterize regional brain tau deposition measured by 18F-AV1451 PET in cognitively normal older adults (CN), mild cognitive impairment (MCI), and AD participants; (2) to evaluate the correlations between cerebrospinal fluid (CSF) biomarkers or Mini-Mental State Examination (MMSE) and 18F-AV1451 PET standardized uptake value ratio (SUVR); and (3) to evaluate the partial volume effects on 18F-AV1451 brain uptake.Methods: The study included total 115 participants (CN = 49, MCI = 58, and AD = 8) from the Alzheimer's Disease Neuroimaging Initiative (ADNI). Preprocessed 18F-AV1451 PET images, structural MRIs, and demographic and clinical assessments were downloaded from the ADNI database. A reblurred Van Cittertiteration method was used for voxelwise partial volume correction (PVC) on PET images. Structural MRIs were used for PET spatial normalization and region of interest (ROI) definition in standard space. The parametric images of 18F-AV1451 SUVR relative to cerebellum were calculated. The ROI SUVR measurements from PVC and non-PVC SUVR images were compared. The correlation between ROI 18F-AV1451 SUVR and the measurements of MMSE, CSF total tau (t-tau), and phosphorylated tau (p-tau) were also assessed.Results:18F-AV1451 prominently specific binding was found in the amygdala, entorhinal cortex, parahippocampus, fusiform, posterior cingulate, temporal, parietal, and frontal brain regions. Most regional SUVRs showed significantly higher uptake of 18F-AV1451 in AD than MCI and CN participants. SUVRs of small regions like amygdala, entorhinal cortex and parahippocampus were statistically improved by PVC in all groups (p < 0.01). Although there was an increasing tendency of 18F-AV-1451 SUVRs in MCI group compared with CN group, no significant difference of 18F-AV1451 deposition was found between CN and MCI brains with or without PVC (p > 0.05). Declined MMSE score was observed with increasing 18F-AV1451 binding in amygdala, entorhinal cortex, parahippocampus, and fusiform. CSF p-tau was positively correlated with 18F-AV1451 deposition. PVC improved the results of 18F-AV-1451 tau deposition and correlation studies in small brain regions.Conclusion: The typical deposition of 18F-AV1451 tau PET imaging in AD brain was found in amygdala, entorhinal cortex, fusiform and parahippocampus, and these regions were strongly associated with cognitive impairment and CSF biomarkers. Although more deposition was observed in MCI group, the 18F-AV-1451 PET imaging could not differentiate the MCI patients from CN population. More tau deposition related to decreased MMSE score and increased level of CSF p-tau, especially in ROIs of amygdala, entorhinal cortex and parahippocampus. PVC did improve the results of tau deposition and correlation studies in small brain regions and suggest to be routinely used in 18F-AV1451 tau PET quantification

Heat stress induced, ligand-independent MET and EGFR signalling in hepatocellular carcinoma

<p><b>Purpose:</b> The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling.</p> <p><b>Materials and Methods:</b> MET⁺ and EGFR⁺ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining.</p> <p><b>Results:</b> Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling.</p> <p><b>Conclusions:</b> Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.</p

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress

<div><p>Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2–3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells <i>in vitro</i> and at the tumor ablation margin <i>in vivo</i>. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.</p></div

Cross comparison of integrated gene expression data from Clone9 rat hepatocyte and N1S1 and AS30D rat HCC cell lines with human HCC (NCI cohort).

<p>Hierarchical clustering analysis of integrated gene expression data from rat cell lines and human HCC tissues. Gene expression data from rat cell lines and human HCC tissues were independently centralized across samples to remove baseline differences between the two data sets. Genes with an expression level at least 2-fold different relative to the median value across tissues in at least 3 tissues in the rat data set were selected for hierarchical clustering analysis (2,776 unique genes). The data are presented in matrix format in which rows represent individual genes and columns represent each tissue. Each cell in the matrix represents the expression level of a gene feature in an individual tissue. The red and green color in cells reflect relative high and low expression levels respectively as indicated in the scale bar (log2 transformed scale). HS, A and B = prognostic subtypes.</p

Representative gross and microscopic pathology and immunohistochemical staining of the ablation zone 24-hours post-ablation in the orthotopic N1S1 HCC model.

<p>(A-B) A) Ablation zone <i>in situ</i> demonstrates a whitish-appearing N1S1 tumor within the liver and a hyperemic rim at the edge of the ablation zone (denoted by white arrowheads). B) Triphenyltetrazolium chloride (TTC) vital staining of cross-section through the ablation zone containing both liver and tumor demonstrates N1S1 tumor within the liver (denoted by white arrowheads) and areas of viable (red) and non-viable (white) tumor and liver within the ablation zone consistent with thermal-ablation induced tissue coagulation. * = laser fiber tract. (C-H) Representative histopathology and phospho-AKT and ERK immunostaining of N1S1 tumors from sham and laser ablation groups. Photomicrographs (100x) of H&E stained sections (C, D) demonstrate C) the liver-tumor margin of a sham-ablated tumor (denoted by white arrowheads) and the D) tumor-ablation margin of a laser ablated tumor (denoted by white arrowheads). Corresponding photomicrographs (100x) of p-AKT (E,F) and p-ERK (G,H) immunostained sections demonstrate markedly increased AKT (F) and ERK (H) phosphorylation at the tumor-ablation margin (denoted by white arrowheads) in the laser-ablated tumor but minimal AKT (E) and ERK (G) phosphorylation in the tumor, background liver or at the tumor-liver margin (denoted by white arrowheads) in the sham-ablated tumor. (*) denotes background liver.</p

Effect of sublethal heat stress on AKT and ERK signaling in HCC cells.

<p>N1S1 and AS30D cells were heat stressed (45°C-10 minutes), recovered up to 2-hours post heat stress and whole-cell lysates were subjected to western immunoblotting for (A and B) phospho- and total AKT and ERK1/2, and (C and D) phospho-BAD, FOXO3a/FOXO1 and GSK3β. β-actin was used as a loading control. Representative images from 1 of 3 independent experiments. BL = baseline, non-heat stress control; t = time post heat stress. For example, t = 0 indicates immediate post-heat stress.</p

Effect of PI3K/mTOR and/or MEK inhibition on heat stress induced AKT, mTOR and ERK signaling and cytotoxicity in HCC cells.

<p>(A) N1S1 and AS30D cells were pre-treated for one-hour with NVP-BEZ235 (0.5μM), AZD6244 (1.0 μM), both or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to western immunoblotting for phospho- and total AKT and ERK, and phospho-mTOR. β-actin was used as a loading control. Representative images from 1 of 3 independent experiments (B) Effect of PI3K/mTOR and/or MEK inhibition on heat stress induced cytotoxicity in HCC cells. N1S1 and AS30D cells pre-treated for one-hour with NVP-BEZ235 (0.1μM), AZD6244 (0.25 or 0.5 μM), both or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) for 10 minutes were assessed using the WST-1 viability assay at 48 hours post-heat stress. Data were normalized to 37°C vehicle control and presented as mean±SEM of 3 independent experiments.</p