Hyperpolarized 13C Magnetic Resonance Imaging (HP-MRI) enables real-time, non-invasive assessment of metabolism in diseases including cancer and neurodegeneration. Broader adoption has been limited by the complexity, duration, and lack of standardization of current hyperpolarization methods. This study evaluated POLARIS Preclinical, a parahydrogen-induced polarization (PHIP) hyperpolarizer designed to streamline production of hyperpolarized 13C agents. Four POLARIS systems were deployed across eight international research centers to produce hyperpolarized [1-13C]pyruvate doses within 90 seconds. In vitro and in vivo imaging was conducted in multiple animal models using MRI systems operating at 1.4, 3, 7, and 9.4 Tesla. Metabolic conversion of pyruvate to lactate and bicarbonate was successfully measured across all sites. POLARIS enabled rapid, reproducible production of hyperpolarized [1-13C]pyruvate and demonstrated consistent performance across instruments, institutions, and field strengths. These results support standardized, high-throughput metabolic MRI for multicenter studies and translational research in oncology, neurology, and cardiovascular disease.

To evaluate hyperpolarized (HP) γ-glutamyl-[1-13C]glycine ([13C]GG) MRI as a non-invasive method for assessing liver injury through imaging of hepatic enzymatic activity. In this prospective preclinical imaging study, five mice underwent HP 13C MRI at baseline and 48 h after induction of acute liver injury with carbon tetrachloride (CCl4). Dynamic slice-selective HP 13C spectra through the liver were acquired at 3 T. Signals from [13C]GG and its cleavage product [1-13C]glycine (catalyzed by γ-glutamyl-transferase or GGT) were quantified. Glycine-to-GG area-under-the-curve (AUC) ratios were calculated. Paired comparisons were analyzed using a two-tailed t-test. Liver histopathology was analyzed after imaging. At baseline, HP [13C]GG demonstrated detectable in vivo hepatic conversion to [1-13C]glycine. Following CCl4-induced injury, glycine production decreased in all animals despite similar substrate delivery. On average, the glycine-to-GG ratio declined by 25

Magnetic resonance imaging of hyperpolarized (HP) [1-13C]pyruvate allows in-vivo assessment of metabolism and has translated into human studies across diseases at 15 centers worldwide. Consensus on best practice for multi-center studies is required to develop clinical applications. This paper presents the results of a 2-round formal consensus building exercise carried out by experts with HP [1-13C]pyruvate human study experience. Twenty-nine participants from 13 sites brought together expertise in pharmacy methods, MR physics, translational imaging, and data-analysis; with the goal of providing recommendations and best practice statements on conduct of multi-center human studies of HP [1-13C]pyruvate MRI. Overall, the group reached consensus on approximately two-thirds of 246 statements in the questionnaire, covering 'HP 13C-Pyruvate Preparation', 'MRI System Setup, Calibration, and Phantoms', 'Acquisition and Reconstruction', and 'Data Analysis and Quantification'. Consensus was present across categories, examples include that: (i) different HP pyruvate preparation methods could be used in human studies, but that the same release criteria have to be followed; (ii) site qualification and quality assurance must be performed with phantoms and that the same field strength must be used, but that the rest of the system setup and calibration methods could be determined by individual sites; (iii) the same pulse sequence and reconstruction methods were preferable, but the exact choice should be governed by the anatomical target; (iv) normalized metabolite area-under-curve (AUC) values and metabolite AUC were the preferred metabolism metrics. The work confirmed areas of consensus for multi-center study conduct and identified where further research is required to ascertain best practice.

Magnetic resonance imaging (MRI) of hyperpolarized (HP) [1-13C]pyruvate is a promising method for measuring cerebral energy metabolism in vivo. The substantial increase in signal provided by HP makes it possible to dynamically monitor the conversion of [1-13C]pyruvate to [1-13C]lactate and [13C]bicarbonate. The HP [1-13C]lactate signal is commonly associated with glycolic activity, whereas [13C]bicarbonate, a by-product of the reaction that forms acetyl-CoA, is linked to oxidative metabolism. However, there is compelling evidence that other factors, such as the concentration of monocarboxylate transporters, influence the production of HP [1-13C]lactate. To clarify the processes responsible for producing the topography of HP [1-13C]pyruvate and its metabolites, we spatially correlated group-average HP 13C MRI images with [18F]FDG, [15O]H2O, [15O]O2, and [15O]CO positron emission topography (PET) images from a separate group of 35 age- and sex-matched adults. We found that [1-13C]pyruvate correlated best with cerebral blood volume (CBV), whereas [1-13C]lactate and [13C]bicarbonate were most strongly associated with cerebral blood flow (CBF), glucose consumption (CMRglc), and oxygen metabolism (CMRO2). Neither [1-13C]lactate nor [13C]bicarbonate was correlated with non-oxidative glucose consumption, also known as aerobic glycolysis. These results are consistent with the view that in the healthy brain, the production of [1-13C]lactate reflects overall energy metabolism rather than being specific to glycolysis.

PurposeAccurate quantification of metabolism in hyperpolarized (HP) 13C MRI is essential for clinical applications. However, kinetic model parameters are often confounded by uncertainties in radiofrequency flip angles and other model parameters.MethodsA data-driven kinetic fitting approach for HP 13C-pyruvate MRI was proposed that compensates for uncertainties in the B1+ field. We hypothesized that introducing a scaling factor to the flip angle to minimize fit residuals would allow more accurate determination of the pyruvate-to-lactate conversion rate (kPL). Numerical simulations were performed under different conditions (flip angle, kPL, and T1 relaxation), with further testing using HP 13C-pyruvate MRI of rat liver and kidneys.ResultsSimulations showed that the proposed method reduced kPL error from 60% to 1% when the prescribed and actual flip angles differed by 60%. The method also showed robustness to T1 uncertainties, achieving median kPL errors within +/- 3% even when the assumed T1 was incorrect by up to a factor of 2. In rat studies, better-quality fitting for lactate signals (a 1.4-fold decrease in root mean square error [RMSE] for lactate fit) and tighter kPL distributions (an average of 3.1-fold decrease in kPL standard deviation) were achieved using the proposed method compared with when no correction was applied.ConclusionThe proposed data-driven kinetic fitting approach provided a method to accurately quantify HP 13C-pyruvate metabolism in the presence of B1+ inhomogeneity. This model may also be used to correct for other error sources, such as T1 relaxation and flow, and may prove to be clinically valuable in improving tumor staging or assessing treatment response.

PURPOSE:The purpose of this study was to show that hyperpolarized (HP) carbon-13 (13C) MRI with multiple co-HP substrates can probe the time course of renal metabolic changes in diabetes. METHODS:[1-13C]pyruvate and [1,3-13C2]acetoacetate were co-HP for simultaneous metabolic assessment of cytosolic and mitochondrial compartments, respectively. A custom multi-band spectral-spatial radiofrequency pulse was designed for enhanced detection of downstream metabolites of both substrates. In vivo co-HP 13C kidney spectra were acquired serially in rats with uncontrolled insulin-deficient diabetes over a period of 8 weeks. Time courses of changes in apparent metabolic conversions of [1-13C]pyruvate and [1,3-13C2]acetoacetate were evaluated and compared with routine clinical markers of kidney disease obtained by serum and urine sampling. RESULTS:Metabolic conversions of both co-HP substrates showed large shifts in diabetic kidney with chronic hyperglycemia. Production of both HP [1-13C]lactate and [1,3-13C2]β-hydroxybutyrate increased over time, with β-hydroxybutyrate signal significantly elevated at 4 weeks, sustained at 8 weeks. Lactate trended higher at 4 weeks, with a larger, significant increase at 8 weeks. Serum and urine markers of renal function were unaltered from baseline throughout the time course, without significant change in serum creatinine nor evidence of albuminuria. CONCLUSION:Noninvasive 13C MRI using multiple co-HP metabolic substrates, whose activities are localized to distinct cellular compartments, could enable early detection of diabetic kidney damage.

Significantly Altered Levels of Proteins Following Treatment with Copanlisib

Apoptotic induction following treatment with eribulin and copanlisib, either alone or in combination by IHC. Representative IHC pictures (A) and quantifications of IHC scores (B) of cleaved PARP on WHIM29 and WHIM34 PDX tumors harvested on day 3 following treatment with either vehicle (day 1), eribulin (day 1), copanlisib (days 1 and 2), or the combination, and FDG PET 4 hours after treatment on day 2.

PURPOSE:To demonstrate hyperpolarization of 15N-caffeine and report exploratory findings as a potential probe of liver function and perfusion. METHODS:An amorphous formulation of [1,3-15N2]caffeine was developed for hyperpolarization via dissolution dynamic nuclear polarization. Polarizer hardware was augmented to support monitoring of solid-state 15N MR signals during the buildup of hyperpolarization. Liquid state hyperpolarized 15N MR signals were obtained in a preclinical 3T magnet by interfacing an external spectrometer console with home-built RF surface coils. 15N signal decay constants were estimated in H2O and in vivo in liver and brain regions of rats at 3 T. Decays were also measured at 9.4 T to assess the effect of B0, and in the presence of albumin to assess the impact of protein binding. RESULTS:Polarization levels of 3.5% and aqueous T1 relaxation times of nearly 200 s were attained for both N1 and N3 positions at 3 T. Shorter apparent decay constants were observed in vivo, ranging from 25 s to 43 s, with modest extensions possible by exploiting competitive binding of iophenoxate with plasma albumin. Downstream products of caffeine could not be detected on in vivo 15N-MR spectra of the liver region, even with metabolic stimulation by β $$ \beta $$ -naphthoflavone treatment. Considering the high perfusion rate of brain, persistence of caffeine signal in this region is consistent with potential value as a perfusion imaging agent. CONCLUSION:These results establish the feasibility of hyperpolarization of hyperpolarized 15N-caffeine, but further work is necessary to establish the role of this new agent to probe liver metabolism and perfusion.

Abstract Non-alcoholic steatohepatitis (NASH) is characterized from its early stages by a profound remodeling of the liver microenvironment, encompassing changes in the composition and activities of multiple cell types and associated gene expression patterns. Hyperpolarized (HP) 13C MRI provides a unique view of the metabolic microenvironment, with potential relevance for early diagnosis of liver disease. Previous studies have detected changes in HP 13C pyruvate to lactate conversion, catalyzed by lactate dehydrogenase (LDH), with experimental liver injury. HP $$\propto $$ ∝ -ketobutyrate ( $$\propto $$ ∝ KB) is a close molecular analog of pyruvate with modified specificity for LDH isoforms, specifically attenuated activity with their LDHA-expressed subunits that dominate liver parenchyma. Building on recent results with pyruvate, we investigated HP $$\propto $$ ∝ KB in methionine-choline deficient (MCD) diet as a model of early-stage NASH. Similarity of results between this new agent and pyruvate (~ 50% drop in cytoplasmic reducing capacity), interpreted together with gene expression data from the model, suggests that changes are mediated through broad effects on intermediary metabolism. Plausible mechanisms are depletion of the lactate pool by upregulation of gluconeogenesis (GNG) and pentose phosphate pathway (PPP) flux, and a possible shift toward increased lactate oxidation. These changes may reflect high levels of oxidative stress and/or shifting macrophage populations in NASH.

Abstract The PI3K pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple-negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the antitumor effect in TNBC. A panel of eight TNBC patient-derived xenograft (PDX) models was tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment-induced signaling changes were examined by reverse phase protein array, immunohistochemistry (IHC) and 18F-fluorodeoxyglucose PET (18F-FDG PET). Compared with each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin-induced mitotic arrest. The combination enhanced induction of apoptosis compared with each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan and AKT phosphorylation by IHC. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin and led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC. Significance: In this research, we demonstrated that the pan-PI3K inhibitor copanlisib enhanced the cytotoxicity of eribulin in a panel of TNBC PDX models. The improved tumor growth inhibition was irrespective of PI3K pathway alteration and was corroborated by the enhanced mitotic arrest and apoptotic induction observed in PDX tumors after combination therapy compared with each drug alone. These data provide the preclinical rationale for the clinical testing in TNBC.

Significantly Altered Proteins Following Treatment with the Combination of Eribulin and Copanlisib including All 8 PDX Models

Tolerability for the Combination of Eribulin and Copanlisib

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of hyperpolarized agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate - by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation, (2) MRI system setup and calibrations, (3) data acquisition and image reconstruction, and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the HP 13C MRI Consensus Group as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods Equipment study groups. It further aims to provide a comprehensive reference for future consensus building as the field continues to advance human studies with this metabolic imaging modality.

PURPOSE:To develop a flexible, vendor-neutral EPI sequence for hyperpolarized 13C metabolic imaging. METHODS:An open-source EPI sequence consisting of a metabolite-specific spectral-spatial RF excitation pulse and a customizable EPI readout was created using the Pulseq framework. To explore the flexibility of our sequence, we tested several versions of the sequence including a symmetric 3D readout with different spatial resolutions for each metabolite (1.0 cm3 and 1.5 cm3). A multichamber phantom constructed with a Shepp-Logan geometry, containing two chambers filled with either natural abundance 13C compounds or hyperpolarized (HP) [1-13C]pyruvate, was used to test each sequence. For experiments involving HP [1-13C]pyruvate, a single chamber was prefilled with nicotinamide adenine dinucleotide hydride and lactate dehydrogenase to facilitate the conversion of [1-13C]pyruvate to [1-13C]lactate. All experiments were performed on a Siemens Prisma 3T scanner. RESULTS:All the sequence variations localized natural-abundance 13C ethylene glycol and methanol to the appropriate compartment of the multichamber phantom. [1-13C]pyruvate was detectable in both chambers following the injection of HP [1-13C]pyruvate, whereas [1-13C]lactate was only found in the chamber containing nicotinamide adenine dinucleotide hydride and lactate dehydrogenase. The conversion rate from [1-13C]pyruvate to [1-13C]lactate (kPL) was 0.01 s-1 (95% confidence interval [0.00, 0.02]). CONCLUSION:We have developed and tested a vendor-neutral EPI sequence for imaging HP 13C agents. We have made all of our sequence creation and image reconstruction code freely available online for other investigators to use.

Expression of protein markers significantly altered by treatment with eribulin in combination with copanlisib in three eribulin-resistant models (WHIM3, WHIM4, and WHIM6). Expression of indicated RPPA protein markers are shown. Aurora_pT288_pT232_pT198, and Histone H3_pS10, were significantly upregulated, with FDR-adjusted P value <0.1, at the completion of 3–4 weeks of treatment with the combination of eribulin and copanlisib versus vehicle. Caspase 7 cleaved was upregulated following combination therapy, although did not reach statistical significance.

Statistical Analysis Comparing Changes in the Levels of Proteins by RPPA Among Different Treatment Groups for 8 PDX Models

PURPOSE:To use the hepatocyte-specific gadolinium-based contrast agent gadoxetate combined with hyperpolarized (HP) [1-13 C]pyruvate MRI to selectively suppress metabolic signals from normal hepatocytes while preserving the signals arising from tumors. METHODS:Simulations were performed to determine the expected changes in HP 13 C MR signal in liver and tumor under the influence of gadoxetate. CC531 colon cancer cells were implanted into the livers of five Wag/Rij rats. Liver and tumor metabolism were imaged at 3 T using HP [1-13 C] pyruvate chemical shift imaging before and 15 min after injection of gadoxetate. Area under the curve for pyruvate and lactate were measured from voxels containing at least 75% of normal-appearing liver or tumor. RESULTS:Numerical simulations predicted a 36% decrease in lactate-to-pyruvate (L/P) ratio in liver and 16% decrease in tumor. In vivo, baseline L/P ratio was 0.44 ± 0.25 in tumors versus 0.21 ± 0.08 in liver (p = 0.09). Following administration of gadoxetate, mean L/P ratio decreased by an average of 0.11 ± 0.06 (p < 0.01) in normal-appearing liver. In tumors, mean L/P ratio post-gadoxetate did not show a statistically significant change from baseline. Compared to baseline levels, the relative decrease in L/P ratio was significantly greater in liver than in tumors (-0.52 ± 0.16 vs. -0.19 ± 0.25, p < 0.05). CONCLUSIONS:The intracellular hepatobiliary contrast agent showed a greater effect suppressing HP 13 C MRI metabolic signals (through T1 shortening) in normal-appearing liver when compared to tumors. The combined use of HP MRI with selective gadolinium contrast agents may allow more selective imaging in HP 13 C MRI.

Tumor growth response to eribulin and copanlisib, either alone or in combination in WHIM29 and WHIM34. A, Tumor volume changes over time compared with that of day 1 after receiving either vehicle, eribulin (0.3 mg/kg i.p. on day 1 of each week × 4, except that 0.1 mg/kg eribulin was administered for the first two doses in WHIM29), copanlisib (10 mg/kg i.v. on days 2 and 3 each week × 4), or the combination of copanlisib and eribulin at the same dosing (n = 5 per group). B, Kaplan–Meier survival duration (days) of tumor-bearing mice after receiving treatments indicated in A. *, P < 0.05; **, P < 0.01, comparing between groups received eribulin or the combination of eribulin and copanlisib.

Treatment induced changes in FDG uptake by PET imaging and in the levels of pAKT by IHC in WHIM29 and WHIM34. Representative images of FDG PET scan of tumor-bearing mice performed pretreatment and posttreatment (on day 2, 4 hours following drug dosing) with either vehicle, copanlisib (10 mg/kg i.v., on day 1, and on day 2), eribulin (0.2 or 0.3 mg/kg i.p., on day 1), or the combination of eribulin and copanlisib for WHIM29 (A) and WHIM34 (B). Quantification of average SUV by FDG PET scan of tumor-bearing mice performed pretreatment and posttreatment with either copanlisib, eribulin, or the combination of copanlisib and eribulin in WHIM29 (n = 5; C) and WHIM34 (n = 5; D). ***, P < 0.001; ****, P < 0.0001. Representative IHC images for pAKT473 and cleaved PARP on tumor tissue sections harvested on day 3, following treatment with either vehicle, copanlisib (10 mg/kg i.v., on day 1, and on day 2), eribulin (0.2 or 0.3 mg/kg i.p., on day 1), or the combination of eribulin and copanlisib for WHIM29 (E) and WHIM34 (F).