Evaluation of \u3csup\u3e18\u3c/sup\u3eF-IAM6067 as a sigma-1 receptor PET tracer for neurodegeneration in vivo in rodents and in human tissue

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. The sigma 1 receptor (S1R) is widely expressed in the CNS and is mainly located on the endoplasmic reticulum. The S1R is involved in the regulation of many neurotransmission systems and, indirectly, in neurodegenerative diseases. The S1R may therefore represent an interesting neuronal biomarker in neurodegenerative diseases such as Parkinson\u27s (PD) or Alzheimer\u27s diseases (AD). Here we present the characterisation of the S1R-specific 18F-labelled tracer 18F-IAM6067 in two animal models and in human brain tissue. Methods: Wistar rats were used for PET-CT imaging (60 min dynamic acquisition) and metabolite analysis (1, 2, 5, 10, 20, 60 min post-injection). To verify in vivo selectivity, haloperidol, BD1047 (S1R ligand), CM398 (S2R ligand) and SB206553 (5HT2B/C antagonist) were administrated for pre-saturation studies. Excitotoxic lesions induced by intra-striatal injection of AMPA were also imaged by 18F-IAM6067 PET-CT to test the sensitivity of the methods in a well-established model of neuronal loss. Tracer brain uptake was also verified by autoradiography in rats and in a mouse model of PD (intrastriatal 6-hydroxydopamine (6-OHDA) unilateral lesion). Finally, human cortical binding was investigated by autoradiography in three groups of subjects (control subjects with Braak ≤2, and AD patients, Braak \u3e2 & ≤4 and Braak \u3e4 stages). Results: We demonstrate that despite rapid peripheral metabolism of 18F-IAM6067, radiolabelled metabolites were hardly detected in brain samples. Brain uptake of 18F-IAM6067 showed differences in S1R anatomical distribution, namely from high to low uptake: pons-raphe, thalamus medio-dorsal, substantia nigra, hypothalamus, cerebellum, cortical areas and striatum. Pre-saturation studies showed 79-90% blockade of the binding in all areas of the brain indicated above except with the 5HT2B/C antagonist SB206553 and S2R ligand CM398 which induced no significant blockade, indicating good specificity of 18F-IAM6067 for S1Rs. No difference between ipsi- and contralateral sides of the brain in the mouse model of PD was detected. AMPA lesion induced a significant 69% decrease in 18F-IAM6067 uptake in the globus pallidus matching the neuronal loss as measured by NeuN, but only a trend to decrease (-16%) in the caudate putamen despite a significant 91% decrease in neuronal count. Moreover, no difference in the human cortical binding was shown between AD groups and controls. Conclusion: This work shows that 18F-IAM6067 is a specific and selective S1R radiotracer. The absence or small changes in S1R detected here in animal models and human tissue warrants further investigations and suggests that S1R might not be the anticipated ideal biomarker for neuronal loss in neurodegenerative diseases such as AD and PD

Spatiotemporal assessment of spontaneous metastasis formation using multimodal in vivo imaging in HER2+ and triple negative metastatic breast cancer xenograft models in mice.

Preclinical breast cancer models recapitulating the clinical course of metastatic disease are crucial for drug development. Highly metastatic cell lines forming spontaneous metastasis following orthotopic implantation were previously developed and characterized regarding their biological and histological characteristics. This study aimed to non-invasively and longitudinally characterize the spatiotemporal pattern of metastasis formation and progression in the MDA-MB-231-derived triple negative LM2-4 and HER2+ LM2-4H2N cell lines, using bioluminescence imaging (BLI), contrast enhanced computed tomography (CT), fluorescence imaging, and 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography ([18F]FDG-PET).LM2-4, LM2-4H2N, and MDA-MB-231 tumors were established in the right inguinal mammary fat pad (MFP) of female SCID mice and resected 14-16 days later. Metastasis formation was monitored using BLI. Metabolic activity of primary and metastatic lesions in mice bearing LM2-4 or LM2-4H2N was assessed by [18F]FDG-PET. Metastatic burden at study endpoint was assessed by CT and fluorescence imaging following intravenous dual-modality liposome agent administration.Comparable temporal metastasis patterns were observed using BLI for the highly metastatic cell lines LM2-4 and LM2-4H2N, while metastasis formed about 10 days later for MDA-MB-231. 21 days post primary tumor resection, metastases were detected in 86% of LM2-4, 69% of LM2-4H2N, and 60% of MDA-MB-231 inoculated mice, predominantly in the axillary region, contralateral MFP, and liver/lung. LM2-4 and LM2-4H2N tumors displayed high metabolism based on [18F]FDG-PET uptake. Lung metastases were detected as the [18F]FDG-PET uptake increased significantly between pre- and post-metastasis scan. Using a liposomal dual-modality agent, CT and fluorescence confirmed BLI detected lesions and identified additional metastatic nodules in the intraperitoneal cavity and lung.The combination of complementary anatomical and functional imaging techniques can provide high sensitivity characterization of metastatic disease spread, progression and overall disease burden. The described models and imaging toolset can be implemented as an effective means for quantitative treatment response evaluation in metastatic breast cancer

<i>In vivo</i> and <i>ex vivo</i> imaging of breast cancer metastasis at study endpoint for LM2-4 and LM2-4H2N.

<p>Imaging of breast cancer metastasis using bioluminescence imaging (BLI), and <i>ex vivo</i> fluorescence and <i>in vivo</i> CT imaging at 48h or 24h, respectively, following injection of the dual-modality contrast agent. Metastatic nodules were confirmed by H&E staining. RA/LA: right/left axillary nodule, P: primary tumor regrowth, L: lung, A: abdominal nodule.</p

Characterization of primary tumor growth for orthotopically implanted LM2-4, LM2-4H2N and MDA-MB-231 cells.

<p>(A) Representative BLI and CT images of primary tumors before resection. Dashed lines outline the tumor contour. R: right. (B) Quantification of primary tumor BLI signal. (C-D) Quantification of primary tumor volume before resection based on CT (C) and caliper measurements (D). (E-G) Correlation between CT tumor volume and BLI signal (E), CT tumor volume and caliper measurements (F), and caliper measurements and BLI signal (G).</p

Spatiotemporal pattern of metastasis formation after removal of the primary LM2-4, LM2-4H2N and MDA-MB-231 tumor.

<p>(A) Representative images of BLI assessment of metastatic burden following removal of the primary LM2-4, LM2-4H2N, and MDA-MB-231 tumor. Arrows illustrate primary regrowth (P) and metastatic regions: left inguinal region (LI), left and right axillary region (LA/RA), liver/lung region (L/L). (B) Metastatic incidences excluding primary regrowth detected by BLI over time after removal of the primary tumor. (C) Metastatic incidences detected by BLI on day 21 post primary tumor resection for different anatomical locations. (D-E) BLI signal from the primary tumor regrowth (D) and from distant metastasis (E) for all animals that eventually develop metastasis. *<i>P</i> < 0.05, <i>t</i>-test. (F) Numbers of animals included in panel D & E.</p

[¹⁸F]FDG-PET of metabolic activity of primary tumors and metastasis of LM2-4 and LM2-4H2N cells.

<p>(A) Representative images of [¹⁸F]FDG uptake in primary tumors of LM2-4 and LM2-4H2N at 11 days post inoculation. Arrows indicate the tumor location. (B) [¹⁸F]FDG uptake in SUV_max for the primary tumor. (C) [¹⁸F]FDG uptake in SUV_max for the muscle. (D) Representative images of [¹⁸F]FDG uptake at different metastatic sites (labelled with arrows) on day 18 post primary tumor removal. (E-F) [¹⁸F]FDG uptake in SUV_max in lung (E) and in metastatic lesions (F) at 18 days post primary tumor removal. (G) Correlation between [¹⁸F]FDG uptake in SUV_max and metastatic nodule volume. L: left. **<i>P</i> < 0.01, ***<i>P</i> < 0.001, Two-way ANOVA/<i>t</i>-test.</p

A systemic transcriptome analysis reveals the regulation of neural stem cell maintenance by an E2F1-miRNA feedback loop.

Stem cell fate decisions are controlled by a molecular network in which transcription factors and miRNAs are of key importance. To systemically investigate their impact on neural stem cell (NSC) maintenance and neuronal commitment, we performed a high-throughput mRNA and miRNA profiling and isolated functional interaction networks of involved mechanisms. Thereby, we identified an E2F1-miRNA feedback loop as important regulator of NSC fate decisions. Although E2F1 supports NSC proliferation and represses transcription of miRNAs from the miR-17 approximately 92 and miR-106a approximately 363 clusters, these miRNAs are transiently up-regulated at early stages of neuronal differentiation. In these early committed cells, increased miRNAs expression levels directly repress E2F1 mRNA levels and inhibit cellular proliferation. In mice, we demonstrated that these miRNAs are expressed in the neurogenic areas and that E2F1 inhibition represses NSC proliferation. The here presented data suggest a novel interaction mechanism between E2F1 and miR-17 approximately 92 / miR-106a approximately 363 miRNAs in controlling NSC proliferation and neuronal differentiation