L-DOS47 Elevates Pancreatic Cancer Tumor pH and Enhances Response to Immunotherapy.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

"Be sustainable": EOSC-Life recommendations for implementation of FAIR principles in life science data handling.

The main goals and challenges for the life science communities in the Open Science framework are to increase reuse and sustainability of data resources, software tools, and workflows, especially in large-scale data-driven research and computational analyses. Here, we present key findings, procedures, effective measures and recommendations for generating and establishing sustainable life science resources based on the collaborative, cross-disciplinary work done within the EOSC-Life (European Open Science Cloud for Life Sciences) consortium. Bringing together 13 European life science research infrastructures, it has laid the foundation for an open, digital space to support biological and medical research. Using lessons learned from 27 selected projects, we describe the organisational, technical, financial and legal/ethical challenges that represent the main barriers to sustainability in the life sciences. We show how EOSC-Life provides a model for sustainable data management according to FAIR (findability, accessibility, interoperability, and reusability) principles, including solutions for sensitive- and industry-related resources, by means of cross-disciplinary training and best practices sharing. Finally, we illustrate how data harmonisation and collaborative work facilitate interoperability of tools, data, solutions and lead to a better understanding of concepts, semantics and functionalities in the life sciences.

Assessing the Therapeutic Efficacy of Proton Transport Inhibitors in a Triple-Negative Breast Cancer Murine Model with Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer Tumor pH Imaging.

Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although few methods can accurately assess their effect in vivo. In this study, we investigated whether MRI-CEST (Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer) tumor pH imaging can be a useful tool to evaluate in vivo the therapeutic efficacy of several Proton Pump Inhibitors (PPIs) in breast cancer. Cell viability and extracellular pH assays were carried out in breast cancer cells cultured at physiological pH (7.4) or acid-adapted (pH of 6.5 and 6.8) following the exposure to inhibitors of V-ATPase (Lansoprazole, Esomeprazole) or NHE1 (Amiloride, Cariporide) at several concentrations. Next, triple-negative breast cancer 4T1 tumor-bearing mice were treated with Lansoprazole or Amiloride and MRI-CEST tumor pH imaging was utilized to assess the in vivo efficacy. Only Lansoprazole induced, in addition to breast cancer cell toxicity, a significant inhibition of proton extrusion. A significant reduction in tumor volume, prolonged survival, and increase in extracellular tumor pH after 1 and 2 weeks were observed after Lansoprazole treatment, whereas no significant changes were detected upon Amiloride treatment. Our results suggested that MRI-CEST tumor pH imaging can monitor the therapeutic efficacy of PPIs in breast cancer murine models.

L-DOS47 enhances response to immunotherapy in pancreatic cancer tumor.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds CEACAM6, a cell surface protein highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Our results demonstrate that combining L DOS47 with anti-PD1 significantly increases the efficacy of anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

In Situ Insonation of Alkaline Buffer Containing Liposomes Leads to a Net Improvement of the Therapeutic Outcome in a Triple Negative Breast Cancer Murine Model.

Breast cancer is characterized by an acidic micro-environment. Acidic extracellular pH gives cancer cells an evolutionary advantage, hence, neutralization of the extracellular pH has been considered as a potential therapeutic strategy. To address the issue of systemic pH alteration, an approach based on the targeted delivery of the buffering solution to the tumor region is investigated. The method relies on the use of low frequency ultrasound and sono-sensitive liposomes loaded with buffers at alkaline pH (LipHUS). After the i.v. injection of LipHUS, the application of ultrasound (US) at the sites of the pathology induces a local increase of pH that results highly effective in i) inhibiting primary tumor growth, ii) reducing tumor recurrence after surgery, and iii) suppressing metastases' formation. The experiments are carried out on a triple negative breast cancer mouse model. The results obtained demonstrate that localized and triggered release of bicarbonate or PBS buffer from sonosensitive liposomes represents an efficient therapeutic tool for treating triple-negative breast cancer. This approach holds promise for potential clinical translation.

Intraperitoneal Delivery of Iopamidol to Assess Extracellular pH of Orthotopic Pancreatic Tumor Model by CEST-MRI.

The extracellular pH (pHe) of solid tumors is often acidic, as a consequence of the Warburg effect, and an altered metabolic state is often associated with malignancy. It has been shown that acidosis can promote tumor progression; thus, many therapeutic strategies have been adopted against tumor metabolism; one of these involves alkalinization therapies to raise tumor pH to inhibit tumor progression, improve immune surveillance, and overcome resistance to chemotherapies. Chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) is a noninvasive technique that can measure pH in vivo using pH-sensitive contrast agents. Iopamidol, an iodinated contrast agent, clinically used for computed tomography (CT), contains amide group protons with pH-dependent exchange rates that can reveal the pHe of the tumor microenvironment. In this study, we optimized intraperitoneal (IP) delivery of iopamidol to facilitate longitudinal assessments of orthotopic pancreatic tumor pHe by CEST-MRI. Following IV-infusion and IP-bolus injections, we compared the two protocols for assessing tumor pH. Time-resolved CT imaging was used to evaluate the uptake of iopamidol in the tumor, revealing that IP-bolus delivered a high amount of contrast agent 40 min postinjection, which was similar to the amounts reached with the IV-infusion protocol. As expected, both IP and IV injection protocols produced comparable measurements of tumor pHe, showing no statistically significant difference between groups (p=0.16). In addition, we showed the ability to conduct longitudinal monitoring of tumor pHe using CEST-MRI with the IP injection protocol, revealing a statistically significant increase in tumor pHe following bicarbonate administration (p < 0.001). In conclusion, this study shows the capability to measure pHe using an IP delivery of iopamidol into orthotopic pancreatic tumors, which is important to conduct longitudinal studies.

Tumor pH Imaging Using Chemical Exchange Saturation Transfer (CEST)-MRI.

Magnetic resonance imaging (MRI) is a noninvasive imaging technique that allows for physiological and functional studies of the tumor microenvironment. Within MRI, the emerging field of chemical exchange saturation transfer (CEST) has been largely exploited for assessing a salient feature of all solid tumors, extracellular acidosis. Iopamidol-based tumor pH imaging has been demonstrated to provide accurate and high spatial resolution extracellular tumor pH maps to elucidate tumor aggressiveness and for assessing response to therapy, with a high potential for clinical translation. Here, we describe the overall setup and steps for measuring tumor extracellular pH of tumor models in mice by exploiting MRI-CEST pH imaging with a preclinical MRI scanner following the administration of iopamidol. We address issues of pH calibration curve setup, animal handling, pH-responsive contrast agent injection, acquisition protocol, and image processing for accurate quantification and visualization of tumor acidosis.

A snapshot of the vast array of diamagnetic CEST MRI contrast agents.

Since the inception of CEST MRI in the 1990s, a number of compounds have been identified as suitable for generating contrast, including paramagnetic lanthanide complexes, hyperpolarized atom cages and, most interesting, diamagnetic compounds. In the past two decades, there has been a major emphasis in this field on the identification and application of diamagnetic compounds that have suitable biosafety profiles for usage in medical applications. Even in the past five years there has been a tremendous growth in their numbers, with more and more emphasis being placed on finding those that can be ultimately used for patient studies on clinical 3 T scanners. At this point, a number of endogenous compounds present in tissue have been identified, and also natural and synthetic organic compounds that can be administered to highlight pathology via CEST imaging. Here we will provide a very extensive snapshot of the types of diamagnetic compound that can generate CEST MRI contrast, together with guidance on their utility on typical preclinical and clinical scanners and a review of the applications that might benefit the most from this new technology.

Di-Pyridine-Containing Macrocyclic Triamide Fe(II) and Ni(II) Complexes as ParaCEST Agents.

Fe(II) and Ni(II) paraCEST contrast agents containing the di-pyridine macrocyclic ligand 2,2',2″-(3,7,10-triaza-1,5(2,6)-dipyridinacycloundecaphane-3,7,10-triyl)triacetamide (DETA) are reported here. Both [Fe(DETA)]2+ and [Ni(DETA)]2+ complexes were structurally characterized. Crystallographic data revealed the seven-coordinated distorted pentagonal bipyramidal geometry of the [Fe(DETA)]·(BF4)2·MeCN complex with five coordinated nitrogen atoms from the macrocyclic ring and two coordinated oxygen atoms from two amide pendant arms. The [Ni(DETA)]·Cl2·2H2O complex was six-coordinated in nature with a distorted octahedral geometry. Four coordinated nitrogen atoms were from the macrocyclic ring, and two coordinated oxygen atoms were from two amide pendant arms. [Fe(DETA)]2+ exhibited well-resolved sharp proton resonances, whereas very broad proton resonances were observed in the case of [Ni(DETA)]2+ due to the long electronic relaxation times. The CEST peaks for the [Fe(DETA)]2+ complex showed one highly downfield-shifted and intense peak at 84 ppm with another shifted but less intense peak at 28 ppm with good CEST contrast efficiency at body temperature, whereas [Ni(DETA)]2+ showed only one highly shifted intense peak at 78 ppm from the bulk water protons. Potentiometric titrations were performed to determine the protonation constants of the ligand and the thermodynamic stability constant of the [M(DETA)]2+ (M = Fe, Co, Ni, Cu, Zn) species at 25.0 °C and I = 0.15 mol·L-1 NaClO4. Metal exchange studies confirmed the stability of the complexes in acidic medium in the presence of physiologically relevant anions and an equimolar concentration of Zn(II) ions.

A Neutral and Stable Macrocyclic Mn(II) Complex for MRI Tumor Visualization.

A stable and inert amphiphilic Mn(II) complex based on a bisamide derivative of 1,4-DO2A (DO2A=tetraazacyclododecane-1,4-diacetic acid) was synthesized and its 1 H NMR relaxometric behavior was investigated as a function of the magnetic field strength, pH and temperature. The interaction with human serum albumin (HSA) was also studied via relaxometry showing a good relaxivity enhancement at low field (at 1T and 298 K the relaxivity increases from 4.5 mM-1 s-1 of the Mn(II)-complex to 14.0 mM-1 s-1 of the complex-HSA supramolecular adduct). In vivo biodistribution and MRI studies highlighted a rapid and mixed renal/liver elimination without spleen accumulation from healthy mice and good contrast enhancing properties in a breast tumor murine model. A comparison with a clinically approved Gd(III) agent (GdBOPTA, Multihance®) underlined that the proposed Mn(II) contrast agent gave comparable tumor contrast enhancement up to 3 hours post-injection.

In Vivo MRI-CEST Tumor pH Imaging Detects Resistance to Proton Pump Inhibitors in Human Prostate Cancer Murine Models.

The tumor microenvironment acidification confers treatment resistance; therefore, the interference with pH regulating systems is considered a new therapeutic strategy. In this study, two human prostate cancer cell lines, PC3 and LNCaP, have been treated in vitro with proton pump inhibitors (PPIs), namely Lansoprazole, Esomeprazole (V-ATPases-inhibitors), Cariporide, and Amiloride (NHE1-inhibitors). The cell viability and pH were assessed at several drug concentrations either at normoxic or hypoxic conditions. Since Esomeprazole showed the highest toxicity towards the PC3 cancer cells compared to LNCaP ones, athymic nude mice bearing subcutaneous or orthotopic PC3 tumors were treated with Esomeprazole (dose: 2.5 mg/kg body weight) for a period of three weeks-and tumor growth was monitored. MRI-CEST tumor pH imaging with Iopamidol was performed upon treatment at 3 h, 1 week (in combination with FDG-PET), and after 2 weeks for evaluating acute, early, and late responses. Although acute tumor pH changes were observed in vivo, long-term studies on both PC3 prostate cancer models did not provide any significant change in tumor acidosis or tumor growth. In conclusion, this work shows that MRI-CEST tumor pH imaging is a valuable tool for assessing the in vivo treatment response to PPIs.

New tools to investigate tumor metabolism by NMR/MRI.

Changes in metabolism is an hallmark that characterizes tumour cells from healthy ones. Their detection can be highly relevant for staging the tumor and for monitoring the response to therapeutic treatments. Herein it is shown the readout of these changes can be achieved either by assessing the pH of the extracellular space in the tumour region and by monitoring real time transformations of hyperpolarized C-13 labelled substrates. Mapping pH in a MR image is possible by measuring the CEST response of an administered contrast agent such as Iopamidol that can provide accurate measurements of the heterogeneity of tumour acidosis. Direct detection of relevant enzymatic activities have been acquired by using Pyruvate and Fumarate hyperpolarized by the incorporation of a molecule of para-H2. Finally, it has been found that the tumour transformation involves an increase in the water exchange rate between the intra- and the extra-cellular compartments. A quantitative estimation of these changes can be obtained by acquiring the longitudinal relaxation times of tissue water protons at low magnetic field strength on Fast Field Cycling Relaxometers. This finding has been exploited in an application devoted to the assessment of the presence of residual tumour tissue in the margins of the resected mass in breast conservative surgery.

XNAT-PIC: Extending XNAT to Preclinical Imaging Centers.

Molecular imaging generates large volumes of heterogeneous biomedical imagery with an impelling need of guidelines for handling image data. Although several successful solutions have been implemented for human epidemiologic studies, few and limited approaches have been proposed for animal population studies. Preclinical imaging research deals with a variety of machinery yielding tons of raw data but the current practices to store and distribute image data are inadequate. Therefore, standard tools for the analysis of large image datasets need to be established. In this paper, we present an extension of XNAT for Preclinical Imaging Centers (XNAT-PIC). XNAT is a worldwide used, open-source platform for securely hosting, sharing, and processing of clinical imaging studies. Despite its success, neither tools for importing large, multimodal preclinical image datasets nor pipelines for processing whole imaging studies are yet available in XNAT. In order to overcome these limitations, we have developed several tools to expand the XNAT core functionalities for supporting preclinical imaging facilities. Our aim is to streamline the management and exchange of image data within the preclinical imaging community, thereby enhancing the reproducibility of the results of image processing and promoting open science practices.

What do we know about dynamic glucose-enhanced (DGE) MRI and how close is it to the clinics? Horizon 2020 GLINT consortium report.

Cancer is one of the most devastating diseases that the world is currently facing, accounting for 10 million deaths in 2020 (WHO). In the last two decades, advanced medical imaging has played an ever more important role in the early detection of the disease, as it increases the chances of survival and the potential for full recovery. To date, dynamic glucose-enhanced (DGE) MRI using glucose-based chemical exchange saturation transfer (glucoCEST) has demonstrated the sensitivity to detect both D-glucose and glucose analogs, such as 3-oxy-methyl-D-glucose (3OMG) uptake in tumors. As one of the recent international efforts aiming at pushing the boundaries of translation of the DGE MRI technique into clinical practice, a multidisciplinary team of eight partners came together to form the "glucoCEST Imaging of Neoplastic Tumors (GLINT)" consortium, funded by the Horizon 2020 European Commission. This paper summarizes the progress made to date both by these groups and others in increasing our knowledge of the underlying mechanisms related to this technique as well as translating it into clinical practice.

GlucoCEST MRI for the Evaluation Response to Chemotherapeutic and Metabolic Treatments in a Murine Triple-Negative Breast Cancer: A Comparison with[18F]F-FDG-PET.

PURPOSE: Triple-negative breast cancer (TNBC) patients have usually poor outcome after chemotherapy and early prediction of therapeutic response would be helpful. [18F]F-FDG-PET/CT acquisitions are often carried out to monitor variation in metabolic activity associated with response to the therapy, despite moderate accuracy and radiation exposure limit its application. The glucoCEST technique relies on the use of unlabelled D-glucose to assess glucose uptake with conventional MRI scanners and is currently under active investigations at clinical level. This work aims at validating the potential of MRI-glucoCEST in monitoring the therapeutic responses in a TNBC tumor murine model. PROCEDURES: Breast tumor (4T1)-bearing mice were treated with doxorubicin or dichloroacetate for 1 week. PET/CT with [18F]F-FDG and MRI-glucoCEST were performed at baseline and after 3 cycles of treatment. Metabolic changes measured with [18F]F-FDG-PET and glucoCEST were compared and evaluated with changes in tumor volumes. RESULTS: Doxorubicin-treated mice showed a significant decrease in tumor growth when compared to the control group. GlucoCEST imaging provided metabolic response after three cycles of treatment. Conversely, no variations were detected in [18F]F-FDG uptake. Dichloroacetate-treated mice did not show any decrease either in tumor volume or in tumor metabolic activity as assessed by both glucoCEST and [18F]F-FDG-PET. CONCLUSIONS: Metabolic changes during doxorubicin treatment can be predicted by glucoCEST imaging that appears more sensitive than [18F]F-FDG-PET in reporting on therapeutic response. These findings support the view that glucoCEST may be a sensitive technique for monitoring metabolic response, but future studies are needed to explore the accuracy of this approach in other tumor types and treatments.

Effect of Esomeprazole Treatment on Extracellular Tumor pH in a Preclinical Model of Prostate Cancer by MRI-CEST Tumor pH Imaging.

Novel anticancer treatments target the pH regulating system that plays a major role in tumor progression by creating an acidic microenvironment, although few studies have addressed their effect on tumor acidosis. In this study, we investigated in vivo several proton pump inhibitors (PPIs) targeting NHE-1 (Amiloride and Cariporide) and V-ATPase (Esomeprazole and Lansoprazole) proton transporters in the DU145 androgen-insensitive human prostate cancer model. In cellulo results showed that DU145 are sensitive, with decreasing efficacy, to Amiloride, Esomeprazole and Lansoprazole, with marked cell toxicity both in normoxia and in hypoxia, with almost any change in pH. In vivo studies were performed upon administration of Esomeprazole to assess both the acute and chronic effects, and Iopamidol-based tumor pH imaging was performed to evaluate tumor acidosis. Although statistically significant tumor pH changes were observed a few hours after Esomeprazole administration in both the acute study and up to one week of treatment in the chronic study, longer treatment resulted in a lack of changes in tumor acidosis, which was associated to similar tumor growth curves between treated and control groups in both the subcutaneous and orthotopic models. Overall, this study highlights MRI-CEST tumor pH imaging as a valid approach to monitoring treatment response to PPIs.

The Release of Inflammatory Mediators from Acid-Stimulated Mesenchymal Stromal Cells Favours Tumour Invasiveness and Metastasis in Osteosarcoma.

Osteosarcoma is the most frequent primary malignant bone tumour with an impressive tendency to metastasise. Highly proliferative tumour cells release a remarkable amount of protons into the extracellular space that activates the NF-kB inflammatory pathway in adjacent stromal cells. In this study, we further validated the correlation between tumour glycolysis/acidosis and its role in metastases. In patients, at diagnosis, we found high circulating levels of inflammatory mediators (IL6, IL8 and miR-136-5p-containing extracellular vesicles). IL6 serum levels significantly correlated with disease-free survival and 18F-FDG PET/CT uptake, an indirect measurement of tumour glycolysis and, hence, of acidosis. In vivo subcutaneous and orthotopic models, co-injected with mesenchymal stromal (MSC) and osteosarcoma cells, formed an acidic tumour microenvironment (mean pH 6.86, as assessed by in vivo MRI-CEST pH imaging). In these xenografts, we enlightened the expression of both IL6 and the NF-kB complex subunit in stromal cells infiltrating the tumour acidic area. The co-injection with MSC also significantly increased lung metastases. Finally, by using 3D microfluidic models, we directly showed the promotion of osteosarcoma invasiveness by acidosis via IL6 and MSC. In conclusion, osteosarcoma-associated MSC react to intratumoural acidosis by triggering an inflammatory response that, in turn, promotes tumour invasiveness at the primary site toward metastasis development.

In vitro and in vivo comparison of MRI chemical exchange saturation transfer (CEST) properties between native glucose and 3-O-Methyl-D-glucose in a murine tumor model.

D-Glucose and 3-O-Methyl-D-glucose (3OMG) have been shown to provide contrast in magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) images. However, a systematic comparison between these two molecules has yet to be performed. The current study deals with the assessment of the effect of pH, saturation power level (B1 ) and magnetic field strength (B0 ) on the MRI-CEST contrast with the aim of comparing the in vivo CEST contrast detectability of these two agents in the glucoCEST procedure. Phosphate-buffered solutions of D-Glucose or 3OMG (20 mM) were prepared at different pH values and Z-spectra were acquired at several B1 levels at 37°C. In vivo glucoCEST images were obtained at 3 and 7 T over a period of 30 min after injection of D-Glucose or 3OMG (at doses of 1.5 or 3 g/kg) in a murine melanoma tumor model (n = 3-5 mice for each molecule, dose and B0 field). A markedly different pH dependence of CEST response was observed in vitro for D-Glucose and 3OMG. The glucoCEST contrast enhancement in the tumor region following intravenous administration (at the 3 g/kg dose) was comparable for both molecules: 1%-2% at 3 T and 2%-3% at 7 T. The percentage change in saturation transfer that resulted was almost constant for 3OMG over the 30-min period, whereas a significant increase was detected for D-Glucose. Our results show similar CEST contrast efficiency but different temporal kinetics for the metabolizable and the nonmetabolizable glucose derivatives in a tumor murine model when administered at the same doses.

Investigating plasma volume expanders as novel macromolecular MRI-CEST contrast agents for tumor contrast-enhanced imaging.

PURPOSE: The aim of this study was to investigate two clinically approved plasma volume expanders (dextran 70 and voluven) as macromolecular MRI-chemical exchange saturation transfer (CEST) contrast agents to assess tumor vascular properties. METHODS: CEST contrast efficiency of both molecules (6% w/v) was measured in vitro at various irradiation saturation powers (1-6 µT for 5 s) and pH values (range, 5.5-7.9) and the exchange rate of hydroxyl protons was calculated. In vivo studies in a murine adenocarcinoma model (n = 4 mice for each contrast agent) upon i.v. injection provided CEST-derived perfusion tumor properties that were compared with those obtained with a gadolinium-based blood-pool agent (Gd-AAZTA-Madec). RESULTS: In vitro measurements showed a marked CEST contrast dependency to pH, with higher CEST contrast at lower pH values for both molecules. The measured prototropic exchange rates confirmed a base-catalyzed exchange rate that was faster for dextran 70 in comparison to voluven. Both molecules showed a similar CEST contrast increase (ΔST% > 3%) in the tumor tissue up to 30 min postinjection, with heterogeneous accumulation. In tumors receiving both CEST and T1 -weighted agents, a voxel-by-voxel analysis indicated moderate spatial correlation of perfusion properties between voluven/dextran 70 and Gd-AAZTA-Madec, suggesting different distribution patterns according to their molecular size. CONCLUSIONS: The obtained results showed that both voluven and dextran 70 can be exploited as MRI-CEST contrast agents for evaluating tumor enhancement properties. Their increased accumulation in tumors and prolonged contrast enhancement promote their use as blood-pool MRI-CEST agents to examine tumor vascularization.