Biology subjects

Prasad, S.

Publications and source records attributed to Prasad, S..

4 recordsLinked to original sources

bioRxiv2019

CMKLR1-targeting peptide tracers for PET/MR imaging of breast cancer

Molecular targeting remains to be a promising approach in cancer medicine. Knowledge about molecular properties such as overexpression of G protein-coupled receptors (GPCRs) is thereby offering a powerful tool for tumor-selective imaging and treatment of cancer cells. We utilized chemerin-based peptides for CMKLR1 receptor targeting in a breast cancer xenograft model. By conjugation with radiolabeled chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of highly specific and affine tracers for hybrid in vivo imaging with positron emission tomography (PET)/ magnetic resonance (MR) and concomitant biodistribution studies.\n\nMethodsWe developed five highly specific and affine peptide tracers targeting CMKLR1 by linker-based conjugation of chemerin peptide analogs (CG34 and CG36) with radiolabeled (68Ga) chelator DOTA. Our established xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. Therefore, we acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments.\n\nResultsThe family of five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values being below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be confirmed by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers 68Ga-DOTA-AHX-CG34, 68Ga-DOTA-KCap-CG34 and 68Ga-DOTA-ADX-CG34 with apparent DU4475 tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 4.5% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. More strikingly, the tumor uptake could be blocked by excess of unlabeled peptide (6.4-fold, 7.2-fold and 3.4-fold 1 h post-injection) and further confirmed the CMKLR1 specificity. As our five tracers, each with particular degree of hydrophobicity, showed different results regarding tumor uptake and organ distribution, we identified these three tracers with moderate, balanced properties to be the most potent in receptor-mediated tumor targeting.\n\nConclusionWith the breast cancer cell line DU4475, we established a model endogenously expressing our target CMKLR1 to evaluate our chemerin-based peptide tracers as highly affine and specific targeting agents. Eventually, we demonstrated the applicability of our 68Ga-labeled tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging and thus developed promising theranostics for tumor treatment.

cancer biology↗

bioRxiv2019

Increasing molar activity by HPLC purification improves 68Ga-DOTA-NAPamide tumor accumulation in a B16/F1 melanoma xenograft model

PurposeMelanocortin receptor 1 is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide. ProceduresThe MC1R ligand DOTA-NAPamide was labeled with 68Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68Ga (95 {degrees}C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. ResultsIn biodistribution studies, the non-purified 68Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and a decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracers for biodistribution. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. ConclusionsWe demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.

cancer biology↗

bioRxiv2019

Predictive markers for Parkinson’s disease using deep neural nets on neuromelanin sensitive MRI

Neuromelanin sensitive magnetic resonance imaging (NMS-MRI) has been crucial in identifying abnormalities in the substantia nigra pars compacta (SNc) in Parkinsons disease (PD). PD is characterized by loss of dopaminergic neurons in SNc and current techniques employ estimation of contrast ratios of the SNc visualized on NMS-MRI to discern PD patients from the healthy controls. However, the extraction of these features is time-consuming and laborious and moreover provides lower prediction accuracies. Furthermore, these do not account for patterns of subtle changes in PD in the SNc. To mitigate this, our work establishes a computer-based analysis technique based on convolutional neural networks (CNNs) to create prognostic and diagnostic biomarkers of PD from NMS-MRI. The technique not only performs with a superior cross-validation accuracy (83.7%) as well as testing accuracy (80%) as compared to contrast ratio-based classification (52.7% cross-validation and 56.5% testing accuracy) and radiomics based classifier (81.1% cross-validation and 60.3% testing accuracy); but also locates the most discriminative regions on the neuromelanin contrast images. These discriminative activations demonstrate that the left SNc plays a key role in the classification in comparison to the right SNc, and are in agreement with the concept of asymmetry in PD. Overall, the proposed technique has the potential to support radiological diagnosis of PD while facilitating deeper understanding into the abnormalities in SNc.

neuroscience↗

bioRxiv2018

Detection of acute TLR-7 agonist-induced hemorrhagic myocarditis in mice by refined multi-parametric quantitative cardiac MRI.

BACKGROUNDHemorrhagic myocarditis is a potentially fatal complication of excessive levels of systemic inflammation. It has been reported in viral infection, but is also possible in systemic autoimmunity. Cardiac magnetic resonance (CMR) imaging is the current gold standard for non-invasive detection of suspected inflammatory damage to the heart and changes in T1 and T2 relaxation times are commonly used to detect edema associated with immune cell infiltration and fibrosis. These measurements also form the basis of the Lake Louise Criteria, which define a framework for the CMR-based diagnosis of myocarditis. However, they do not take into account the possibility of hemorrhage leading to tissue iron deposition which strongly influences T1 and T2 measurements and may complicate interpretation based on these two parameters only.\n\nMETHODSSystemic inflammation was induced in CFN mice by application of the TLR-7 agonist Resiquimod. Histopathology was performed on heart sections to assess immune cell infiltration (Hematoxylin & Eosin), fibrosis (PicoSirius Red) and tissue iron deposition (Perls Prussian Blue). A multi-parametric cardiac MRI tissue mapping approach measuring T1, T2, and T2* relaxation times was established to non-invasively identify these parameters in this small rodent model.\n\nRESULTSResiquimod-treated mice developed severe thrombocytopenia and hemorrhagic myocarditis. We identified patches of cardiac hemorrhage based on the presence of two major MRI phenotypes. Increased T2 with normal T1 and T2* values correlated with infiltration/edema only, while decreased T1, T2, and T2* values identify areas with infiltration/edema in the presence of iron indicating hemorrhagic myocarditis.\n\nCONCLUSIONWe show that over-activation of the TLR-7 pathway by Resiquimod treatment of CFN mice induces an early immune response reminiscent of excessive systemic inflammation due to viral infection. This causes internal bleeding which manifests most prominently as severe hemorrhagic myocarditis. We optimized a comprehensive noninvasive in vivo MRI approach based on quantitative measurement of T1, T2 and T2* to demonstrate the presence of diffuse myocardial edema, infiltration and iron deposition without the need of contrast agent administration. We propose that adding quantitative T2* mapping to CMR protocols for detection of myocarditis will improve diagnostic sensitivity and interpretation of disease mechanisms.

physiology↗