Natural pH regulatory mechanisms can be overruled during several pathologies such as cancer, inflammation and ischaemia, leading to local pH changes in the human body. Here we demonstrate that ¹³C-labelled zymonic acid (ZA) can be used as hyperpolarized magnetic resonance pH imaging sensor. ZA is synthesized from [1-¹³C]pyruvic acid and its ¹³C resonance frequencies shift up to 3.0 p.p.m. per pH unit in the physiological pH range. The long lifetime of the hyperpolarized signal enhancement enables monitoring of pH, independent of concentration, temperature, ionic strength and protein concentration. We show in vivo pH maps within rat kidneys and subcutaneously inoculated tumours derived from a mammary adenocarcinoma cell line and characterize ZA as non-toxic compound predominantly present in the extracellular space. We suggest that ZA represents a reliable and non-invasive extracellular imaging sensor to localize and quantify pH, with the potential to improve understanding, diagnosis and therapy of diseases characterized by aberrant acid-base balance.

Metabolic magnetic resonance imaging (MRI) using hyperpolarized (HP) pyruvate is becoming a non‐invasive technique for diagnosing, staging, and monitoring response to treatment in cancer and other diseases. The clinically established method for producing HP pyruvate, dissolution dynamic nuclear polarization, however, is rather complex and slow. Signal Amplification By Reversible Exchange (SABRE) is an ultra‐fast and low‐cost method based on fast chemical exchange. Here, for the first time, we demonstrate not only in vivo utility, but also metabolic MRI with SABRE. We present a novel routine to produce aqueous HP [1‐¹³C]pyruvate‐d₃ for injection in 6 minutes. The injected solution was sterile, non‐toxic, pH neutral and contained ≈30 mM [1‐¹³C]pyruvate‐d₃ polarized to ≈11 % (residual 250 mM methanol and 20 μM catalyst). It was obtained by rapid solvent evaporation and metal filtering, which we detail in this manuscript. This achievement makes HP pyruvate MRI available to a wide biomedical community for fast metabolic imaging of living organisms.

Urea transporter can be used as a sensitive, contrast agent-free gene reporter for magnetic resonance imaging in cells and whole animals. Non-invasive imaging of gene expression can be used to track implanted cells in vivo but often requires the addition of an exogenous contrast agent that may have limited tissue access1. We show that the urea transporter (UT-B) can be used as a gene reporter, where reporter expression is detected using 1H MRI measurements of UT-B-mediated increases in plasma membrane water exchange. HEK cells transfected with the reporter showed an increased apparent water exchange rate (AXR), which increased in line with UT-B expression. AXR values measured in vivo, in UT-B-expressing HEK cell xenografts, were significantly higher (about twofold, P < 0.0001), compared with non-expressing controls. Fluorescence imaging of a red fluorescent protein (mStrawberry), co-expressed with UT-B showed that UT-B expression correlated in a linear fashion with AXR. Transduction of rat brain cells in situ with a lentiviral vector expressing UT-B resulted in about a twofold increase in AXR at the site of virus injection.

The PDCD1-encoded immune checkpoint receptor PD-1 is a key tumor suppressor in T cells that is recurrently inactivated in T cell non-Hodgkin lymphomas (T-NHLs). The highest frequencies of PDCD1 deletions are detected in advanced disease, predicting inferior prognosis. However, the tumor-suppressive mechanisms of PD-1 signaling remain unknown. Here, using tractable mouse models for T-NHL and primary patient samples, we demonstrate that PD-1 signaling suppresses T cell malignancy by restricting glycolytic energy and acetyl coenzyme A (CoA) production. In addition, PD-1 inactivation enforces ATP citrate lyase (ACLY) activity, which generates extramitochondrial acetyl-CoA for histone acetylation to enable hyperactivity of activating protein 1 (AP-1) transcription factors. Conversely, pharmacological ACLY inhibition impedes aberrant AP-1 signaling in PD-1-deficient T-NHLs and is toxic to these cancers. Our data uncover genotype-specific vulnerabilities in PDCD1-mutated T-NHL and identify PD-1 as regulator of AP-1 activity.