White light emission from helically stacked humin-mimic based H-aggregates in heteroatom free carbon dots.

White light emission (WLE), particularly from heteroatom free carbon dots (CDs), is unusual. Besides, deciphering the origin of WLE from a H-aggregated molecular fluorophore in such kinds of CDs is a challenging task due to their non-fluorescent character resulting from a forbidden transition from a lower-energy excitonic state. Therefore, rigorous investigation on their elusive excited state photophysical properties along with their steady-state optical phenomena has to be carried out to shed light on the nature of distinct emissive states formed in the CDs. Herein, for the first time, we report WLE from imperfect H-aggregates of co-facially π-π stacked humin-like structures comprising furfural monomer units as a unique molecular fluorophore in CDs, as revealed from combined spectroscopic and microscopic studies, synthesized through hydrothermal treatment of the single precursor, dextrose. H-aggregates in CDs show a broad range of excitation-dependent emission spectra with color coordinates close to pure white light, i.e., CIE (0.35, 0.37) and a color temperature of 6000 K. Imperfect orientation between the transition dipole moments of adjacent monomer units in the H-aggregate's molecular arrangement is expected to cause ground state symmetry breaking, as confirmed by Circular Dichroism (CD) studies, which established helically stacked nature in molecular aggregates and produced significant oscillatory strength at lower energy excitonic states to enable fluorescence. TRES and TAS investigations have been performed to minimise the intricacies associated with excited state photophysics, which is regarded as an essential step in gaining a grasp on emissive states. Based on the observation of two isoemissive spots in the time-resolved area normalized emission spectra (TRANES), the existence of three oligomeric species in the excited state equilibrium of the pure/hybrid H-aggregates has been established. The exciton dynamics through electron relaxation from the higher to the lower excitonic states, charge transfer (CT) states, and surface trap mediated emission in excimer states of H-aggregates have also been endorsed as three distinct emissive states from femtosecond transient absorption spectroscopy (TAS) studies corroborating with their steady-state absorption and emission behavior. The results would demonstrate the usage of CDs as a cutting-edge fluorescent material for creating aggregate-induced white light emission.

Exploiting metabolic vulnerabilities after anti-VEGF antibody therapy in ovarian cancer.

Despite modest clinical improvement with anti-vascular endothelial growth factor antibody (AVA) therapy in ovarian cancer, adaptive resistance is ubiquitous and additional options are limited. A dependence on glutamine metabolism, via the enzyme glutaminase (GLS), is a known mechanism of adaptive resistance and we aimed to investigate the utility of a GLS inhibitor (GLSi). Our in vitro findings demonstrated increased glutamine abundance and a significant cytotoxic effect in AVA-resistant tumors when GLSi was administered in combination with bevacizumab. In vivo, GLSi led to a reduction in tumor growth as monotherapy and when combined with AVA. Furthermore, GLSi initiated after the emergence of resistance to AVA therapy resulted in a decreased metabolic conversion of pyruvate to lactate as assessed by hyperpolarized magnetic resonance spectroscopy and demonstrated robust antitumor effects with a survival advantage. Given the increasing population of patients receiving AVA therapy, these findings justify further development of GLSi in AVA resistance.

Metabolic Imaging Using Hyperpolarization for Assessment of Premalignancy.

There is an unmet need for noninvasive surrogate markers that can help identify premalignant lesions across different tumor types. Here we describe the methodology and technical details of protocols employed for in vivo 13C pyruvate metabolic imaging experiments. The goal of the method described is to identify and understand metabolic changes, to enable detection of pancreatic premalignant lesions, as a proof of concept of the high sensitivity of this imaging modality.

Hyperpolarized Magnetic Resonance and Artificial Intelligence: Frontiers of Imaging in Pancreatic Cancer.

BACKGROUND: There is an unmet need for noninvasive imaging markers that can help identify the aggressive subtype(s) of pancreatic ductal adenocarcinoma (PDAC) at diagnosis and at an earlier time point, and evaluate the efficacy of therapy prior to tumor reduction. In the past few years, there have been two major developments with potential for a significant impact in establishing imaging biomarkers for PDAC and pancreatic cancer premalignancy: (1) hyperpolarized metabolic (HP)-magnetic resonance (MR), which increases the sensitivity of conventional MR by over 10,000-fold, enabling real-time metabolic measurements; and (2) applications of artificial intelligence (AI). OBJECTIVE: Our objective of this review was to discuss these two exciting but independent developments (HP-MR and AI) in the realm of PDAC imaging and detection from the available literature to date. METHODS: A systematic review following the PRISMA extension for Scoping Reviews (PRISMA-ScR) guidelines was performed. Studies addressing the utilization of HP-MR and/or AI for early detection, assessment of aggressiveness, and interrogating the early efficacy of therapy in patients with PDAC cited in recent clinical guidelines were extracted from the PubMed and Google Scholar databases. The studies were reviewed following predefined exclusion and inclusion criteria, and grouped based on the utilization of HP-MR and/or AI in PDAC diagnosis. RESULTS: Part of the goal of this review was to highlight the knowledge gap of early detection in pancreatic cancer by any imaging modality, and to emphasize how AI and HP-MR can address this critical gap. We reviewed every paper published on HP-MR applications in PDAC, including six preclinical studies and one clinical trial. We also reviewed several HP-MR-related articles describing new probes with many functional applications in PDAC. On the AI side, we reviewed all existing papers that met our inclusion criteria on AI applications for evaluating computed tomography (CT) and MR images in PDAC. With the emergence of AI and its unique capability to learn across multimodal data, along with sensitive metabolic imaging using HP-MR, this knowledge gap in PDAC can be adequately addressed. CT is an accessible and widespread imaging modality worldwide as it is affordable; because of this reason alone, most of the data discussed are based on CT imaging datasets. Although there were relatively few MR-related papers included in this review, we believe that with rapid adoption of MR imaging and HP-MR, more clinical data on pancreatic cancer imaging will be available in the near future. CONCLUSIONS: Integration of AI, HP-MR, and multimodal imaging information in pancreatic cancer may lead to the development of real-time biomarkers of early detection, assessing aggressiveness, and interrogating early efficacy of therapy in PDAC.

Melanoma Evolves Complete Immunotherapy Resistance through the Acquisition of a Hypermetabolic Phenotype.

Despite the clinical success of T-cell checkpoint blockade, most patients with cancer still fail to have durable responses to immunotherapy. The molecular mechanisms driving checkpoint blockade resistance, whether preexisting or evolved, remain unclear. To address this critical knowledge gap, we treated B16 melanoma with the combination of CTLA-4, PD-1, and PD-L1 blockade and a Flt3 ligand vaccine (≥75% curative), isolated tumors resistant to therapy, and serially passaged them in vivo with the same treatment regimen until they developed complete resistance. Using gene expression analysis and immunogenomics, we determined the adaptations associated with this resistance phenotype. Checkpoint resistance coincided with acquisition of a "hypermetabolic" phenotype characterized by coordinated upregulation of the glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways. These resistant tumors flourished under hypoxic conditions, whereas metabolically starved T cells lost glycolytic potential, effector function, and the ability to expand in response to immunotherapy. Furthermore, we found that checkpoint-resistant versus -sensitive tumors could be separated by noninvasive MRI imaging based solely on their metabolic state. In a cohort of patients with melanoma resistant to both CTLA-4 and PD-1 blockade, we observed upregulation of pathways indicative of a similar hypermetabolic state. Together, these data indicated that melanoma can evade T-cell checkpoint blockade immunotherapy by adapting a hypermetabolic phenotype.

Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease.

Therapeutic efficacy of liposomal Grb2 antisense oligodeoxynucleotide (L-Grb2) in preclinical models of ovarian and uterine cancer.

BACKGROUND: Adaptor proteins such as growth factor receptor-bound protein-2 (Grb2) play important roles in cancer cell signaling. In the present study, we examined the biological effects of liposomal antisense oligodeoxynucleotide that blocks Grb2 expression (L-Grb2) in gynecologic cancer models. MATERIALS AND METHODS: Murine orthotopic models of ovarian (OVCAR5 and SKOV3ip1) and uterine (Hec1a) cancer were used to study the biological effects of L-Grb2 on tumor growth. In vitro experiments (cell viability assay, Western blot analysis, siRNA transfection, and reverse phase protein array) were carried out to elucidate the mechanisms and potential predictors of tumor response to L-Grb2. FINDINGS: Treatment with L-Grb2 decreased tumor growth and metastasis in orthotopic models of ovarian cancer (OVCAR5, SKOV3ip1) by reducing angiogenesis and increasing apoptosis at a dose of 15 mg/kg with no effect on mouse body weight. Treatment with L-Grb2 and paclitaxel led to the greatest decrease in tumor weight (mean ± SEM, 0.17 g ± 0.10 g) compared with that in control mice (0.99 g ± 0.35 g). We also observed a reduction in tumor burden after treatment with L-Grb2 and the anti-VEGF antibody B-20 (86% decrease in tumor weight compared with that in controls). Ovarian cancer cells with ErbB2 amplification (OVCAR8 and SKOV3ip1) were the most sensitive to Grb2 downregulation. Reverse phase protein array analysis identified significant dysregulation of metabolites (LDHA, GAPDH, and TCA intermediates) in ovarian cancer cells after Grb2 downregulation. INTERPRETATION: L-Grb2 has therapeutic efficacy in preclinical models of ovarian and uterine cancer. These findings support further clinical development of L-Grb2.

Early Detection of Pancreatic Intraepithelial Neoplasias (PanINs) in Transgenic Mouse Model by Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy.

While pancreatic cancer (PC) survival rates have recently shown modest improvement, the disease remains largely incurable. Early detection of pancreatic cancer may result in improved outcomes and therefore, methods for early detection of cancer, even premalignant lesions, may provide more favorable outcomes. Pancreatic intraepithelial neoplasias (PanINs) have been identified as premalignant precursor lesions to pancreatic cancer. However, conventional imaging methods used for screening high-risk populations do not have the sensitivity to detect PanINs. Here, we have employed hyperpolarized metabolic imaging in vivo and nuclear magnetic resonance (1H-NMR) metabolomics ex vivo to identify and understand metabolic changes, towards enabling detection of early PanINs and progression to advanced PanINs lesions that precede pancreatic cancer formation. Progression of disease from tissue containing predominantly low-grade PanINs to tissue with high-grade PanINs showed a decreasing alanine/lactate ratio from high-resolution NMR metabolomics ex vivo. Hyperpolarized magnetic resonance spectroscopy (HP-MRS) allows over 10,000-fold sensitivity enhancement relative to conventional magnetic resonance. Real-time HP-MRS was employed to measure non-invasively changes of alanine and lactate metabolites with disease progression and in control mice in vivo, following injection of hyperpolarized [1-13C] pyruvate. The alanine-to-lactate signal intensity ratio was found to decrease as the disease progressed from low-grade PanINs to high-grade PanINs. The biochemical changes of alanine transaminase (ALT) and lactate dehydrogenase (LDH) enzyme activity were assessed. These results demonstrate that there are significant alterations of ALT and LDH activities during the transformation from early to advanced PanINs lesions. Furthermore, we demonstrate that real-time conversion kinetic rate constants (kPA and kPL) can be used as metabolic imaging biomarkers of pancreatic premalignant lesions. Findings from this emerging HP-MRS technique can be translated to the clinic for detection of pancreatic premalignant lesion in high-risk populations.

Assessing Metabolic Intervention with a Glutaminase Inhibitor in Real-Time by Hyperpolarized Magnetic Resonance in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematopoietic disease characterized by glutamine-dependent metabolism. A novel glutaminase (GLS) inhibitor, CB-839, is currently under evaluation for treatment of hematopoietic malignancies and solid tumors. Our purpose was to measure cellular changes in AML associated with CB-839 treatment and to test the ability of hyperpolarized pyruvate for interrogating these changes to OCI-AML3 cells. Our results show that treatment with CB-839 interfered with the citric acid cycle, reduced the NADH/NAD+ ratio and ATP levels, reduced cell proliferation and viability, and reduced the basal and maximal respiratory capacities [oxygen consumption rate (OCR)]. We observed a reduction of the conversion of hyperpolarized pyruvate to lactate in cell lines and in a mouse AML model after CB-839 treatment. Our in vitro and in vivo results support the hypothesis that, in AML, glutamine is utilized to generate reducing equivalents (NADH, FADH2) through the citric acid cycle and that reduction in redox state by GLS inhibition decreases the rate of pyruvate to lactate conversion catalyzed by lactate dehydrogenase. We propose hyperpolarized pyruvate/lactate measurement as a method for direct monitoring of metabolic changes occurring in AML patients receiving CB-839. With further optimization, this method may provide a noninvasive imaging tool to assess the early efficacy of therapeutic intervention with GLS inhibitors.

Combining Hyperpolarized Real-Time Metabolic Imaging and NMR Spectroscopy To Identify Metabolic Biomarkers in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer that progresses without any symptom, and oftentimes, it is detected at an advanced stage. The lack of prior symptoms and effective treatments have created a knowledge gap in the management of this lethal disease. This issue can be addressed by developing novel noninvasive imaging-based biomarkers in PDAC. We explored in vivo hyperpolarized (HP) 13C MRS of pyruvate to lactate conversion and ex vivo 1H NMR spectroscopy in a panel of well-annotated patient-derived PDAC xenograft (PDXs) model and investigated the correlation between aberrant glycolytic metabolism and aggressiveness of the tumor. Real-time metabolic imaging data demonstrate the immediate intracellular conversion of HP 13C pyruvate to lactate after intravenous injection interrogating upregulated lactate dehydrogenase (LDH) activity in aggressive PDXs. Total ex vivo lactate measurement by 1H NMR spectroscopy showed a direct correlation with in vivo dynamic pyruvate-to-lactate conversion and demonstrated the potential of dynamic metabolic flux as a biomarker of total lactate concentration and aggressiveness of the tumor. Furthermore, the metabolite concentrations were very distinct among all four tumor types analyzed in this study. Overexpression of LDH-A and hypoxia-inducible factor (HIF-1α) plays a significant role in the conversion kinetics of HP pyruvate-to-lactate in tumors. Collectively, these data identified aberrant metabolic characteristics of pancreatic cancer PDXs and could potentially delineate metabolic targets for therapeutic intervention. Metabolic imaging with HP pyruvate and NMR metabolomics may enable identification and classification of aggressive subtypes of patient-derived xenografts. Translation of this real-time metabolic technique to the clinic may have the potential to improve the management of patients at high risk of developing pancreatic diseases.

Assessing Therapeutic Efficacy in Real-time by Hyperpolarized Magnetic Resonance Metabolic Imaging.

Precisely measuring tumor-associated alterations in metabolism clinically will enable the efficient assessment of therapeutic responses. Advances in imaging technologies can exploit the differences in cancer-associated cell metabolism as compared to normal tissue metabolism, linking changes in target metabolism to therapeutic efficacy. Metabolic imaging by Positron Emission Tomography (PET) employing 2-fluoro-deoxy-glucose ([18F]FDG) has been used as a routine diagnostic tool in the clinic. Recently developed hyperpolarized Magnetic Resonance (HP-MR), which radically increases the sensitivity of conventional MRI, has created a renewed interest in functional and metabolic imaging. The successful translation of this technique to the clinic was achieved recently with measurements of 13C-pyruvate metabolism. Here, we review the potential clinical roles for metabolic imaging with hyperpolarized MRI as applied in assessing therapeutic intervention in different cancer systems.

Androgen Receptor Signaling in Castration-Resistant Prostate Cancer Alters Hyperpolarized Pyruvate to Lactate Conversion and Lactate Levels In Vivo.

PURPOSE: Androgen receptor (AR) signaling affects prostate cancer (PCa) growth, metabolism, and progression. Often, PCa progresses from androgen-sensitive to castration-resistant prostate cancer (CRPC) following androgen-deprivation therapy. Clinicopathologic and genomic characterizations of CRPC tumors lead to subdividing CRPC into two subtypes: (1) AR-dependent CRPC containing dysregulation of AR signaling alterations in AR such as amplification, point mutations, and/or generation of splice variants in the AR gene; and (2) an aggressive variant PCa (AVPC) subtype that is phenotypically similar to small cell prostate cancer and is defined by chemotherapy sensitivity, gain of neuroendocrine or pro-neural marker expression, loss of AR expression, and combined alterations of PTEN, TP53, and RB1 tumor suppressors. Previously, we reported patient-derived xenograft (PDX) animal models that contain characteristics of these CRPC subtypes. In this study, we have employed the PDX models to test metabolic alterations in the CRPC subtypes. PROCEDURES: Mass spectrometry and nuclear magnetic resonance analysis along with in vivo hyperpolarized 1-[13C]pyruvate spectroscopy experiments were performed on prostate PDX animal models. RESULTS: Using hyperpolarized 1-[13C]pyruvate conversion to 1-[13C]lactate in vivo as well as lactate measurements ex vivo, we have found increased lactate production in AR-dependent CRPC PDX models even under low-hormone levels (castrated mouse) compared to AR-negative AVPC PDX models. CONCLUSIONS: Our analysis underscores the potential of hyperpolarized metabolic imaging in determining the underlying biology and in vivo phenotyping of CRPC.

Prostate Cancer Energetics and Biosynthesis.

Cancers must alter their metabolism to satisfy the increased demand for energy and to produce building blocks that are required to create a rapidly growing tumor. Further, for cancer cells to thrive, they must also adapt to an often changing tumor microenvironment, which can present new metabolic challenges (ex. hypoxia) that are unfavorable for most other cells. As such, altered metabolism is now considered an emerging hallmark of cancer. Like many other malignancies, the metabolism of prostate cancer is considerably different compared to matched benign tissue. However, prostate cancers exhibit distinct metabolic characteristics that set them apart from many other tumor types. In this chapter, we will describe the known alterations in prostate cancer metabolism that occur during initial tumorigenesis and throughout disease progression. In addition, we will highlight upstream regulators that control these metabolic changes. Finally, we will discuss how this new knowledge is being leveraged to improve patient care through the development of novel biomarkers and metabolically targeted therapies.

Metabolic Differences in Glutamine Utilization Lead to Metabolic Vulnerabilities in Prostate Cancer.

The new oncologic paradigm of precision medicine is focused on identifying metabolic, proteomic, transcriptomic and genomic variabilities in tumors that can be exploited to tailor treatments and improve patient outcomes. Metabolic changes are a hallmark of cancer, and inhibition of metabolic pathways is now a major strategy in medicinal chemistry for targeting cancers. However, non-invasive biomarkers to categorize metabolic subtypes are in short supply. The purpose of this study was to characterize the intracellular and extracellular metabolic profiles of four prostate cancer cell lines with varying degrees of aggressiveness. We observed metabolic differences between the aggressive prostate cancer cell line PC3 and the even more aggressive, metastatic subline PC3M assessed by hyperpolarized in vivo pyruvate studies, nuclear magnetic resonance spectroscopy, and carbon-13 feeding studies. On further examination of the differences between these two cell lines, we found increased glutamine utilization in the metastatic PC3M subline that led directly to sensitivity to glutaminase inhibitor CB-839. Our study supports the theory that metastatic progression increases glutamine utilization and the inhibition of glutaminolysis could have clinical implications.

Interrogating Metabolism in Brain Cancer.

This article reviews existing and emerging techniques of interrogating metabolism in brain cancer from well-established proton magnetic resonance spectroscopy to the promising hyperpolarized metabolic imaging and chemical exchange saturation transfer and emerging techniques of imaging inflammation. Some of these techniques are at an early stage of development and clinical trials are in progress in patients to establish the clinical efficacy. It is likely that in vivo metabolomics and metabolic imaging is the next frontier in brain cancer diagnosis and assessing therapeutic efficacy; with the combined knowledge of genomics and proteomics a complete understanding of tumorigenesis in brain might be achieved.

Therapeutic Targeting of the Warburg Effect in Pancreatic Cancer Relies on an Absence of p53 Function.

The "Warburg effect" describes a peculiar metabolic feature of many solid tumors, namely their increased glucose uptake and high glycolytic rates, which allow cancer cells to accumulate building blocks for the biosynthesis of macromolecules. During aerobic glycolysis, pyruvate is preferentially metabolized to lactate by the enzyme lactate dehydrogenase-A (LDH-A), suggesting a possible vulnerability at this target for small-molecule inhibition in cancer cells. In this study, we used FX11, a small-molecule inhibitor of LDH-A, to investigate this possible vulnerability in a panel of 15 patient-derived mouse xenograft (PDX) models of pancreatic cancer. Unexpectedly, the p53 status of the PDX tumor determined the response to FX11. Tumors harboring wild-type (WT) TP53 were resistant to FX11. In contrast, tumors harboring mutant TP53 exhibited increased apoptosis, reduced proliferation indices, and attenuated tumor growth when exposed to FX11. [18F]-FDG PET-CT scans revealed a relative increase in glucose uptake in mutant TP53 versus WT TP53 tumors, with FX11 administration downregulating metabolic activity only in mutant TP53 tumors. Through a noninvasive quantitative assessment of lactate production, as determined by 13C magnetic resonance spectroscopy (MRS) of hyperpolarized pyruvate, we confirmed that FX11 administration inhibited pyruvate-to-lactate conversion only in mutant TP53 tumors, a feature associated with reduced expression of the TP53 target gene TIGAR, which is known to regulate glycolysis. Taken together, our findings highlight p53 status in pancreatic cancer as a biomarker to predict sensitivity to LDH-A inhibition, with regard to both real-time noninvasive imaging by 13C MRS as well as therapeutic response.

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Monitoring chemotherapeutic response by hyperpolarized 13C-fumarate MRS and diffusion MRI.

Targeted chemotherapeutic agents often do not result in tumor shrinkage, so new biomarkers that correlate with clinical efficacy are needed. In this study, we investigated noninvasive imaging protocols to monitor responses to sorafenib, a multikinase inhibitor approved for treatment of renal cell and hepatocellular carcinoma. Healthy cells are impermeable to fumarate, so conversion of this metabolite to malate as detected by (13)C-magnetic resonance spectroscopy (MRS) has been suggested as one marker for cell death and treatment response in tumors. Diffusion MRI also has been suggested as a measure of therapy-induced cytotoxic edema because viable cells act as a diffusion barrier in tissue. For these reasons, we assessed sorafenib responses using hyperpolarized (13)C-fumarate, diffusion-weighted MRI (DW-MRI) in a xenograft model of human breast cancer in which daily administration of sorafenib was sufficient to stabilize tumor growth. We detected signals from fumarate and malate following intravenous administration of hyperpolarized fumarate with a progressive increase in the malate-to-fumarate (MA/FA) ratio at days 2 to 5 after sorafenib infusion. The apparent diffusion coefficient (ADC) measured by DW-MRI increased in the treated group consistent with cytotoxic edema. However, the MA/FA ratio was a more sensitive marker of therapeutic response than ADC, with 2.8-fold versus 1.3-fold changes, respectively, by day 5 of drug treatment. Histologic analyses confirmed cell death in the sorafenib-treated cohort. Notably, (13)C-pyruvate-to-lactate conversion was not affected by sorafenib in the breast cancer model examined. Our results illustrate how combining hyperpolarized substrates with DW-MRI can allow noninvasive monitoring of targeted therapeutic responses at relatively early times after drug administration.

Nanodiamond as a New Hyperpolarizing Agent and Its (13)C MRS.

In this work, we have hyperpolarized carbonaceous nanoparticles (D ≈ 10 nm), that is, "nanodiamonds", with 1.1% (13)C (natural abundance) using dynamic nuclear polarization (DNP). The polarization buildup curve showed a signal enhancement with relative intensity up to 4700 at 1.4 K and 100 mW microwave power. (13)C magnetic resonance spectra (MRS) were obtained from the sample at 7 T, and the signal decayed with a T1 of 55 ± 3s. Notably, polarization was possible in the absence of added radical, consistent with previous results showing endogenous unpaired electrons in natural nanodiamonds. These likely contribute to the shorter T1's compared to those of highly pure diamond. Despite the relatively short T1, these observations suggest that natural nanodiamonds may be useful for in vivo applications.

Evaluation of LDH-A and glutaminase inhibition in vivo by hyperpolarized 13C-pyruvate magnetic resonance spectroscopy of tumors.

Hyperpolarized (13)C magnetic resonance spectroscopy provides a unique opportunity to detect real-time metabolic fluxes as a means to measure metabolic treatment responses in vivo. Here, we show that pharmacologic inhibition of lactate dehydrogenase-A suppressed the conversion of hyperpolarized (13)C-pyruvate to lactate in murine xenografts of P493 human lymphoma. In contrast, a glutaminase inhibitor reduced conversion of (13)C-pyruvate to alanine without affecting conversion of pyruvate to lactate. These results illustrate the ability to monitor biomarkers for responses to antimetabolic therapy in real-time, paving the way for clinical development of imaging biomarkers to monitor metabolic pharmacodynamics.