Lactate plays a critical role in the tumor microenvironment, driving tumor progression, metastasis, and immune evasion. Despite its importance, in vivo quantification of lactate using magnetic resonance spectroscopy (MRS) has faced challenges, primarily due to the overlapping lipid signal at 1.3 ppm. Current clinical practice employs a long echo time to exploit differences in T2 relaxation between lactate and lipids; however, this approach significantly suppresses signals from other metabolites. Lipid has a notably different T1 relaxation time than lactate and other metabolites, which may be exploited by an inversion recovery sequence to better distinguish them. However, this method has not found wide use because of the loss of signal in other metabolites. Here, we introduce a selective inversion pulse with a short echo time MRS method (SPIR-PRESS), which mitigates this issue. In phantom experiments, SPIR-PRESS successfully suppressed lipid signals that could be misinterpreted as lactate in short TE PRESS spectra, while maintaining sensitivity to the full metabolite profile. SPIR-PRESS demonstrated superior performance in quantifying lactate compared to long echo time PRESS, with ~60% increase in sensitivity for lactate detection compared to conventional PRESS with a 288-ms TE. In a mouse glioma model, SPIR-PRESS clearly detected lactate and other key tumor metabolites (total choline, creatine, NAA) in the tumor, which were not detectable in conventional long TE PRESS. These findings highlight SPIR-PRESS as a promising technique for improved lactate quantification and comprehensive metabolite profiling in tumor environments.

We investigated the effects of senescence and hypoxia on the transcriptome and secretome of the colon cancer cell, HCT-116, in an in vitro model. Senescence was confirmed using SA-β Gal staining and the expression of p53 and p21 proteins, and hypoxia using HIF-1α protein. Control (CN) and senescent (SN) cells were exposed to normoxia or hypoxia, control hypoxia (CH), and senescent hypoxia (SH). Senescence (SN, SH) enhanced the expression of kallikrein-related peptidases, TPp53, p21, optineurin, lipocalin, ADH-1, and stratifin by several folds. Stratifin, with tumor suppressive functions, was upregulated in senescent cells under normoxia but not in hypoxia. Hypoxia (CH and SH) upregulated the expression of many glycolysis genes, especially HK, PFK, aldolase, PDH kinase, and LDH-A. Mitochondrial RNAs (tRNA and rRNA) were increased in SH compared to CH. Significant increases in the secretion of IL-1α, endothelin, bFGF, HB-EGF, PDGF-AB, CCL-5, 7, 22, and CXCL-1 and 8 were observed in SN and SH. VEGF-A, VEGF-C, and TNF-β secretion increased, while PLGF, TGF-α, IL-27, GM-CSF, and M-CSF decreased under hypoxic (CH and SH) conditions. Thus, senescence and hypoxia contribute to cancer cell senescence pathophysiology by regulating the cellular transcriptome and secretome and by both positive and negative feedback mechanisms.

Abstract Patients with hereditary leiomyomatosis and renal cell carcinoma (HLRCC), characterized by mutations in the fumarate hydratase (FH) gene, are at risk for development of aggressive FH-deficient RCCs. FH-deficient tumor cells undergo a pronounced and irreversible metabolic shift to lactate fermentation due in part to loss and mutation of mitochondrial DNA. Fumarate accumulation in FH-deficient tumor cells leads to increased expression of NAD(P)H-quinone oxidoreductase 1 (NQO1) through activation of the NRF2 transcription factor. Although several therapeutic agents have shown promise in the treatment of FH-deficient RCC, clinical outcomes in patients remain unsatisfactory. In this study, we examined the mechanism and therapeutic efficacy of isobutyl-deoxynyboquinone (IB-DNQ), which undergoes futile redox cycling in the presence of NQO1 and oxygen, leading to sustained generation of the highly reactive and toxic superoxide anion. First, we found that patient-derived FH-deficient tumor cells exhibit minimal oxygen consumption in vitro, and EPR oxygen mapping of FH-deficient tumor xenografts in vivo revealed that tumor oxygen levels were elevated relative to other genetically defined in vivo models of RCC. Infusion of IB-DNQ in tumor-bearing animals resulted in rapid and robust non-mitochondrial oxygen consumption in FH-deficient tumor xenografts as measured by both EPR oxygen imaging and photoacoustic mapping of tumor hemoglobin saturation. Repeated doses of IB-DNQ resulted in reduced tumor growth rates. Metabolomic analyses revealed that IB-DNQ treatment strongly suppressed glycolysis and reduced cellular ATP levels by rapidly depleting NADH and NADPH in FH-deficient tumor cells. Finally, [1-13C]pyruvate hyperpolarized MR spectroscopy revealed decreased conversion of pyruvate to lactate in FH-deficient tumor xenografts following IB-DNQ treatment, providing a direct measurement of the impact of IB-DNQ on lactate fermentation in vivo. The combination of these in vivo imaging techniques and metabolite measurements demonstrate that NQO1-activated quinones can effectively target aerobic glycolysis in FH-deficient tumors which rely heavily on lactate fermentation for growth. Citation Format: Yuki Shibata, Shun Kishimoto, Ye Yang, Ming-Hui Wei, Julia Medina-Velazquez, Burchelle Blackman, Jeeva Munasinghe, Viraj Chegu, Vaishnavi S. Srirama, Tyler A. On, Nallathamby Devasahayam, Chandramouli V. Gadisetti, Jeffrey R. Brender, Murali C. Krishna, Daniel R. Crooks, William Marston Linehan. EPR imaging of oxygen consumption driven by NQO1-activated compounds in FH-deficient renal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7328.

Hyperpolarized (HP) carbon-13 [13C] enables the specific investigation of dynamic metabolic and physiologic processes via in vivo MRI-based molecular imaging. As the leading HP metabolic agent, [1-13C]pyruvate plays a pivotal role due to its rapid tissue uptake and central role in cellular energetics. Dissolution dynamic nuclear polarization (d-DNP) is considered the gold standard method for the production of HP metabolic probes; however, development of a faster, less expensive technique could accelerate the translation of metabolic imaging via HP MRI to routine clinical use. Signal Amplification by Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) achieves rapid hyperpolarization by using parahydrogen (p-H2) as the source of nuclear spin order. Currently, SABRE is clinically limited due to the toxicity of the iridium catalyst, which is crucial to the SABRE process. To mitigate Ir contamination, we introduce a novel iteration of the SABRE catalyst, incorporating bis(polyfluoroalkylated) imidazolium salts. This novel perfluorinated SABRE catalyst retained polarization properties while exhibiting an enhanced hydrophobicity. This modification allows the easy removal of the perfluorinated SABRE catalyst from HP [1-13C]-pyruvate after polarization in an aqueous solution, using the ReD-SABRE protocol. The residual Ir content after removal was measured via ICP-MS at 177 ppb, which is the lowest reported to date for pyruvate and is sufficiently safe for use in clinical investigations. Further improvement is anticipated once automated processes for delivery and recovery are initiated. SABRE-SHEATH using the perfluorinated SABRE catalyst can become an attractive low-cost alternative to d-DNP to prepare biocompatible HP [1-13C]-pyruvate formulations for in vivo applications in next-generation molecular imaging modalities.

Aminopeptidases (APs) in the renin-angiotensin system (RAS) and their activity balance play crucial roles in regulating vascular functions. Multiplexed analysis of RAS-related AP activities is useful for diagnosing diseases including cancer. Dynamic nuclear polarization-coupled magnetic resonance imaging (DNP-MRI) enables the simultaneous detection of multiple enzymatic activities in vivo. However, developing practical DNP-MRI probes, especially for multiplexed detection, remains challenging. Here, we report the design of DNP-MRI probes for the in vivo multiplexed analysis of AP activities. By integrating quantum mechanical calculations, organic synthesis, and physicochemical and biochemical evaluations, we developed a series of AP-responsive DNP-MRI probes with high enzymatic reactivities and distinguishable chemical shifts. Using these probes, we successfully detected and visualized multiple AP activities in vivo. Furthermore, we performed in vivo multiplexed analysis of RAS-related AP activities in tumor-bearing mice, demonstrating the potential of this approach for monitoring the efficacy of antiangiogenic cancer therapy and for the accurate discrimination of tumor types.

Real-time invasion analysis of UOK262 and UOK365 cells treated with OT-82. Real-time invasion analysis of UOK262 and UOK365 cells demonstrating cell line invasion over 5 days (120 hrs) was performed (A) to show that DMSO (used as the vehicle for OT-82) did not affect invasion in comparison to untreated cells and (B) to demonstrate the effects of OT-82 treatment at either 1 nM or 10 nM in comparison to vehicle alone (DMSO) or the non-invasive control (no serum in the lower chamber). In each case, a representative graph from one of three separate experiments is shown. (C) The invasion inhibition for three repeats at each dose is shown for each cell line. The average invasion inhibition is shown as a percentage compared to the vehicle alone (DMSO) invasion and T-tests were performed comparing each dose to the vehicle alone (DMSO).

Supplemental Figure S1: Effect of NAMPT inhibition and evaluation of NAD+ producing enzymes in a panel of molecularly diverse RMS cell lines and models

Supplemental Table S2: Dose range testing and IC50 parameters for OT-82 in RMS cell lines

Background/Objectives: Thousands of nephrectomies are performed annually in the United States, but the short-term metabolic effects of surgically induced renal ischemia remain unclear. The conventional metabolic markers used to characterize post-surgical renal function, such as creatinine and GFR, are measured in the serum but do not provide metabolic information about the renal parenchyma itself. We aimed to characterize the immediate metabolic effects of surgical ischemia on renal parenchyma within a temporal framework. Methods: Timed renal parenchyma biopsies were collected from eight patients undergoing nephrectomy for renal cell carcinoma both prior to and after ligation of the renal hilum. These samples were ground, extracted, and analyzed using nuclear magnetic resonance (NMR) spectroscopy to measure changes in lactate, succinate, glucose, alanine, and glycine levels. Results: Due to experimental limitations, we were only able to draw limited conclusions from three patients. Of the five remaining patients, all had significant increases in lactate and succinate levels as a function of time, though the degree to which these increases occurred varied between each patient. Glucose levels generally decreased in the renal parenchyma but did not necessarily correlate with lactate production, assuming all glucose underwent fermentation to lactate in a hypoxic environment. Alanine and glycine levels did not change in a predictable pattern across patients. Conclusions: There are significant changes in lactate, glucose and succinate levels within minutes of the onset of renal ischemia in human patients. The degree of change in the metabolites analyzed varied significantly between patients. The length of surgical ischemia must be considered during surgical procurement of tumor specimens for metabolomic analysis.

In vitro analysis of NAMPT inhibition on glycolysis in FH-deficient tumor cells. (A) Effects of the NAMPT inhibitor OT-82 (100 nM) on the extracellular acidification rate (ECAR) in the UOK262 and UOK365 HLRCC cell lines and the RPTEC normal cell line. (B) Representation of the different effects of OT-82 versus DMSO at time point 5 (designated by a star) after the injection of glucose in UOK262, UOK365, and RPTEC. 2-DG, 2-deoxyglucose.

Pharmacokinetic profile of OT-82 in nude and NSG mice. (A) Bioanalysis of plasma concentrations of OT-82 were made using a validated LC-MS/MS assay (n=3 nude mice, 50 mg/kg, 7 intervals; n=3 NSG mice, 75mg/kg, 7 intervals). (B) Pharmacokinetic parameters were calculated after a single dose of OT-82. The data from all mice in each group were pooled together to assume one “average” mouse per group.

Proliferation analysis of UOK268 and RPTEC in response to OT-82 and nicotinic acid rescue of NAD+/NADH depletion. (A-B) UOK268 or RPTEC cells were grown in 96 well plates from an initial plating of 2,000 cells and cellular confluency was monitored in real-time by an Incucyte S3 Live-Cell Imaging System. After 24 hours of initial measurements cells were treated with either DMSO, 1 nM OT-82, 5 nM OT-82, 10 nM OT-82, or 10 nM OT-82 plus 1 mM of nicotinamide mononucleotide (NMN) and evaluated for an additional 5 days. (C-D) The effect of NMN on NAD+/NADH depletion by OT-82 in UOK268 and RPTEC cells is shown. (E) Effects on the levels of NAD+ and NADH after 24 hours of treatment with either 10nM OT-82, or 10nM OT-82 in combination with 1 mM nicotinic acid (NA) in UOK262, UOK268, and UOK365.

Supplemental Table S1: Molecular features of RMS cell lines

Renal oxygenation is essential for maintaining kidney function. Disruptions in oxygen delivery can lead to renal hypoxia, which can exacerbate kidney injury through multiple pathways, including inflammation, oxidative stress, and ischemia-reperfusion injury. Despite the recognized importance of oxygenation in renal pathology, noninvasive and reliable methods for assessing kidney oxygen levels are limited. Current techniques either lack sensitivity or involve invasive procedures, restricting their use in routine monitoring. Therefore, there is a pressing need for innovative approaches to map renal oxygenation, particularly in kidney injury. This study evaluated electron paramagnetic resonance (EPR)-based oxygen imaging using the paramagnetic tracer Ox071 to map kidney oxygen levels in mice with cyclophosphamide-induced kidney injury. Urine partial pressure of oxygen (Po2) was also assessed as a potential surrogate marker. EPR oximetry accurately measured kidney oxygen distribution, revealing a temporary increase in Po2 post-injury. Urine oximetry, however, did not reliably reflect changes in kidney oxygenation. Furthermore, EPR oximetry provided high-resolution spatial mapping of oxygen levels within the kidney, allowing for a detailed understanding of the impact of hypoxia on renal tissue. EPR oximetry is a promising, noninvasive tool for monitoring renal oxygenation, offering high-resolution mapping and longitudinal assessment. Its ability to provide detailed information about oxygen distribution within the kidney makes it a valuable tool for studying the pathophysiology of renal diseases and for developing novel therapeutic strategies.NEW & NOTEWORTHY Quantitative spatially resolved measurement of renal oxygenation has the potential to guide clinical decision making in renal disorders such as acute kidney injury. In this study, we demonstrate the utility of electron paramagnetic resonance imaging to provide noninvasive and quantitative high-resolution mapping of kidney oxygen concentrations.

Validation studies to evaluate inhibition of additional HLRCC cell lines by NAMPT inhibitors. A) Effects of the NAMPT inhibitor GNE-618 on cell viability in FH -/- HLRCC cells lines UOK348, UOK271, UOK350, UOK268, UOK365, FH restored UOK268WT and non-transformed kidney epithelial cells RPTEC. Cell viability was assessed by Cell Titer-Glo assay at 96 h. B) Effects of the NAMPT inhibitor OT-82 on cell viability in FH -/- HLRCC cells lines UOK348, UOK271, UOK350, UOK268, UOK365, FH restored UOK268WT and non-transformed kidney epithelial cells RPTEC.

NAMPT immunohistochemical analysis in HLRCC tumors and normal kidney. (A) In patient #1, both a primary kidney HLRCC tumor (upper panel) and an associated metastatic mass (lower panel) demonstrated strong NAMPT staining. Surrounding non-tumor tissues show little NAMPT staining. (B) In patient #4, a primary kidney HLRCC tumor (upper panel) shows strong NAMPT staining in comparison to normal kidney tissue present on the same slide (lower panel). (C) In patient #5, a metastatic mass shows strong NAMPT staining (upper panel). Material from this mass was used to derive the UOK262 cell line, which shows similar positive staining when grown as a xenograft (lower panel). (D) In patient #6, a primary kidney HLRCC tumor demonstrated strong NAMPT staining. Matching H&E staining is included for all samples (A-D).

Supplemental Figure S6: Gene set enrichment analysis (GSEA) comparing RMS cell lines that undergo a non-necrotic response to OT-82 versus a necrotic response to OT-82

The abscopal effect, where localized radiation therapy induces regression of distant metastatic lesions through immune activation, shows promise for treating metastatic cancer but occurs inconsistently. Here we demonstrate that tumor perfusion critically influences systemic immune responses to combination therapy with radiation and PD-1 blockade. Using multimodal imaging including DCE-MRI, EPR oximetry, and hyperpolarized 13C-MRI, we show that successful abscopal responses in MC38 tumors are characterized by enhanced perfusion, reduced hypoxia, decreased cellularity, and lower glycolytic activity in remote tumors. Notably, pre-treatment perfusion metrics (AUC1min) and extracellular volume (AUC10min) in primary tumors predict subsequent growth of remote tumors, while the same measurements in remote tumors lack predictive value. Based on these findings, we enhanced the abscopal effect by exposing mice to carbogen (95% O2 + 5% CO2) during radiation therapy. Carbogen exposure increased tumor perfusion by 71% (AUC1min) and significantly improved systemic responses in the checkpoint blockade responsive MC38 model but not in the poorly responsive B16.F10 tumors. The enhanced response correlated with increased activation of CD8+ T cells in tumor-draining lymph nodes and elevated serum HMGB-1 levels. RNA sequencing revealed significant extracellular matrix remodeling in carbogen-treated tumors. These results establish tumor perfusion as both a predictive biomarker and a modifiable determinant of systemic immune responses, suggesting that perfusion-based patient stratification and vascular modification strategies could improve outcomes in combination immunotherapy and radiation treatment.