Multiparametric chemical exchange saturation transfer MRI detects metabolic changes in breast cancer following immunotherapy.

BACKGROUND: With metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy. METHODS: Tumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTRasym) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry. RESULTS: During tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models. CONCLUSION: Multiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition.

Vascular response patterns to targeted therapies in murine breast cancer models with divergent degrees of malignancy.

BACKGROUND: Response assessment of targeted cancer therapies is becoming increasingly challenging, as it is not adequately assessable with conventional morphological and volumetric analyses of tumor lesions. The tumor microenvironment is particularly constituted by tumor vasculature which is altered by various targeted therapies. The aim of this study was to noninvasively assess changes in tumor perfusion and vessel permeability after targeted therapy in murine models of breast cancer with divergent degrees of malignancy. METHODS: Low malignant 67NR or highly malignant 4T1 tumor-bearing mice were treated with either the multi-kinase inhibitor sorafenib or immune checkpoint inhibitors (ICI, combination of anti-PD1 and anti-CTLA4). Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with i.v. injection of albumin-binding gadofosveset was conducted on a 9.4 T small animal MRI. Ex vivo validation of MRI results was achieved by transmission electron microscopy, immunohistochemistry and laser ablation-inductively coupled plasma-mass spectrometry. RESULTS: Therapy-induced changes in tumor vasculature differed between low and highly malignant tumors. Sorafenib treatment led to decreased tumor perfusion and endothelial permeability in low malignant 67NR tumors. In contrast, highly malignant 4T1 tumors demonstrated characteristics of a transient window of vascular normalization with an increase in tumor perfusion and permeability early after therapy initiation, followed by decreased perfusion and permeability parameters. In the low malignant 67NR model, ICI treatment also mediated vessel-stabilizing effects with decreased tumor perfusion and permeability, while ICI-treated 4T1 tumors exhibited increasing tumor perfusion with excessive vascular leakage. CONCLUSION: DCE-MRI enables noninvasive assessment of early changes in tumor vasculature after targeted therapies, revealing different response patterns between tumors with divergent degrees of malignancy. DCE-derived tumor perfusion and permeability parameters may serve as vascular biomarkers that allow for repetitive examination of response to antiangiogenic treatment or immunotherapy.

Multiparametric MRI enables for differentiation of different degrees of malignancy in two murine models of breast cancer.

Objective: The objective of this study was to non-invasively differentiate the degree of malignancy in two murine breast cancer models based on identification of distinct tissue characteristics in a metastatic and non-metastatic tumor model using a multiparametric Magnetic Resonance Imaging (MRI) approach. Methods: The highly metastatic 4T1 breast cancer model was compared to the non-metastatic 67NR model. Imaging was conducted on a 9.4 T small animal MRI. The protocol was used to characterize tumors regarding their structural composition, including heterogeneity, intratumoral edema and hemorrhage, as well as endothelial permeability using apparent diffusion coefficient (ADC), T1/T2 mapping and dynamic contrast-enhanced (DCE) imaging. Mice were assessed on either day three, six or nine, with an i.v. injection of the albumin-binding contrast agent gadofosveset. Ex vivo validation of the results was performed with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), histology, immunhistochemistry and electron microscopy. Results: Significant differences in tumor composition were observed over time and between 4T1 and 67NR tumors. 4T1 tumors showed distorted blood vessels with a thin endothelial layer, resulting in a slower increase in signal intensity after injection of the contrast agent. Higher permeability was further reflected in higher Ktrans values, with consecutive retention of gadolinium in the tumor interstitium visible in MRI. 67NR tumors exhibited blood vessels with a thicker and more intact endothelial layer, resulting in higher peak enhancement, as well as higher maximum slope and area under the curve, but also a visible wash-out of the contrast agent and thus lower Ktrans values. A decreasing accumulation of gadolinium during tumor progression was also visible in both models in LA-ICP-MS. Tissue composition of 4T1 tumors was more heterogeneous, with intratumoral hemorrhage and necrosis and corresponding higher T1 and T2 relaxation times, while 67NR tumors mainly consisted of densely packed tumor cells. Histogram analysis of ADC showed higher values of mean ADC, histogram kurtosis, range and the 90th percentile (p90), as markers for the heterogenous structural composition of 4T1 tumors. Principal component analysis (PCA) discriminated well between the two tumor models. Conclusions: Multiparametric MRI as presented in this study enables for the estimation of malignant potential in the two studied tumor models via the assessment of certain tumor features over time.

Multiparametric Magnetic Resonance Imaging for Immediate Target Hit Assessment of CD13-Targeted Tissue Factor tTF-NGR in Advanced Malignant Disease.

Early assessment of target hit in anti-cancer therapies is a major task in oncologic imaging. In this study, immediate target hit and effectiveness of CD13-targeted tissue factor tTF-NGR in patients with advanced malignant disease enrolled in a phase I trial was assessed using a multiparametric MRI protocol. Seventeen patients with advanced solid malignancies were enrolled in the trial and received tTF-NGR for at least one cycle of five daily infusions. Tumor target lesions were imaged with multiparametric MRI before therapy initiation, five hours after the first infusion and after five days. The imaging protocol comprised ADC, calculated from DWI, and DCE imaging and vascular volume fraction (VVF) assessment. DCE and VVF values decreased within 5 h after therapy initiation, indicating early target hit with a subsequent decrease in tumor perfusion due to selective tumor vessel occlusion and thrombosis induced by tTF-NGR. Simultaneously, ADC values increased at five hours after tTF-NGR administration. In four patients, treatment had to be stopped due to an increase in troponin T hs, with subsequent anticoagulation. In these patients, a reversed effect, with DCE and VVF values increasing and ADC values decreasing, was observed after anticoagulation. Changes in imaging parameters were independent of the mean vessel density determined by immunohistochemistry. By using a multiparametric imaging approach, changes in tumor perfusion after initiation of a tumor vessel occluding therapy can be evaluated as early as five hours after therapy initiation, enabling early assessment of target hit.

First-In-Class CD13-Targeted Tissue Factor tTF-NGR in Patients with Recurrent or Refractory Malignant Tumors: Results of a Phase I Dose-Escalation Study.

BACKGROUND: Aminopeptidase N (CD13) is present on tumor vasculature cells and some tumor cells. Truncated tissue factor (tTF) with a C-terminal NGR-peptide (tTF-NGR) binds to CD13 and causes tumor vascular thrombosis with infarction. METHODS: We treated 17 patients with advanced cancer beyond standard therapies in a phase I study with tTF-NGR (1-h infusion, central venous access, 5 consecutive days, and rest periods of 2 weeks). The study allowed intraindividual dose escalations between cycles and established Maximum Tolerated Dose (MTD) and Dose-Limiting Toxicity (DLT) by verification cohorts. RESULTS: MTD was 3 mg/m2 tTF-NGR/day × 5, q day 22. DLT was an isolated and reversible elevation of high sensitivity (hs) Troponin T hs without clinical sequelae. Three thromboembolic events (grade 2), tTF-NGR-related besides other relevant risk factors, were reversible upon anticoagulation. Imaging by contrast-enhanced ultrasound (CEUS) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) showed major tumor-specific reduction of blood flow in all measurable lesions as proof of principle for the mode of action of tTF-NGR. There were no responses as defined by Response Evaluation Criteria in Solid Tumors (RECIST), although some lesions showed intratumoral hemorrhage and necrosis after tTF-NGR application. Pharmacokinetic analysis showed a t1/2(terminal) of 8 to 9 h without accumulation in daily administrations. CONCLUSION: tTF-NGR is safely applicable with this regimen. Imaging showed selective reduction of tumor blood flow and intratumoral hemorrhage and necrosis.

Investigations into modeling and further estimation of detection limits of the liquid electrode dielectric barrier discharge.

The liquid electrode dielectric barrier discharge (LE-DBD) is a miniaturized atmospheric pressure plasma as emission excitation source for elemental determination with pulsed behavior. Metals dissolved in liquids are detectable in flow systems with low flow rates of 20 µL min(-1) by means of optical emission spectrometry using a simple portable spectrometer. Time-resolved determination of the hydrogen excitation temperature Tαß indicates that the LE-DBD does not reach a stable state during a burning phase, whereat the maximum and minimum Tαß is independent of the flow rate. Adding dissolved metals to the liquid electrode does not influence the minimum Tαß at the end of a burning phase. With the help of measured doubly charged lanthanum lines and spatially resolved measurements, the mechanism of the liquid transfer into the plasma will be clarified. Emissions from metal oxides indicate a thermal evaporation transfer mechanism, but only an additional electrospray-like transfer mechanism can explain the observed La III emissions and nonhomogeneous spatial distribution of exited species. The reaction pathways for electrosprayed hydrated metal ions are discussed for triply and doubly charged ions. The analytical performance is evaluated for 23 elements from the categories of alkali, alkaline earth, transition, and poor metals. The achieved detection limits are between 0.016 mg L(-1) for Li and 41 mg L(-1) for Bi.

The influence of the ionizer geometry on the absolute density calibration of reactive neutral species in a molecular beam mass spectrometry.

Molecular beam mass spectrometry is a powerful diagnostic technique, which can be used for the measurement of absolute number densities of reactive species in non-equilibrium reactive plasmas. However, the calibration of absolute number densities is susceptible to systematic errors. Critical issues are the proper design of the sampling system and the correction of the background signal. Here we discuss the effect of reflections of particles from the molecular beam in an ionizer, formation of additional background particle density in the ionizer, and its effect on the density calibration of reactive particle densities. A Monte Carlo simulation of particle trajectories in the ionizer is used to estimate the detection probability of a beam particle after the collision with the ionizer wall. The simulation shows that as much as two-third of the signal can be due to scattered particles in the commercially available mass spectrometers. This effect leads to systematic underestimation of densities of reactive particles, which are reactive at the surface and, therefore, do not have any background density. A simple change in the ionizer geometry is suggested, which can significantly reduce this problem.