PFKFB3-mediated Pro-glycolytic Shift in Hepatocellular Carcinoma Proliferation.

BACKGROUND & AIMS: Metabolic reprogramming, in particular, glycolytic regulation, supports abnormal survival and growth of hepatocellular carcinoma (HCC) and could serve as a therapeutic target. In this study, we sought to identify glycolytic regulators in HCC that could be inhibited to prevent tumor progression and could also be monitored in vivo, with the goal of providing a theragnostic alternative to existing therapies. METHODS: An orthotopic HCC rat model was used. Tumors were stimulated into a high-proliferation state by use of off-target liver ablation and were compared with lower-proliferating controls. We measured in vivo metabolic alteration in tumors before and after stimulation, and between stimulated tumors and control tumors using hyperpolarized 13C magnetic resonance imaging (MRI) (h13C MRI). We compared metabolic alterations detected by h13C MRI to metabolite levels from ex vivo mass spectrometry, mRNA levels of key glycolytic regulators, and histopathology. RESULTS: Glycolytic lactate flux increased within HCC tumors 3 days after tumor stimulation, correlating positively with tumor proliferation as measured with Ki67. This was associated with a shift towards aerobic glycolysis and downregulation of the pentose phosphate pathway detected by mass spectrometry. MRI-measured lactate flux was most closely coupled with PFKFB3 expression and was suppressed with direct inhibition using PFK15. CONCLUSIONS: Inhibition of PFKFB3 prevents glycolytic-mediated HCC proliferation, trackable by in vivo h13C MRI.

Hyperpolarized [1-13C]Pyruvate Magnetic Resonance Spectroscopic Imaging for Evaluation of Early Response to Tyrosine Kinase Inhibition Therapy in Gastric Cancer.

PURPOSE: To evaluate the use of hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging (HP-13C MRSI) for quantitative measurement of early changes in glycolytic metabolism and its ability to predict response to pan-tyrosine kinase inhibitor (Pan-TKI) therapy in gastric cancer (GCa). PROCEDURES: Pan-TKI afatinib-sensitive NCI-N87 and resistant SNU16 human GCa cells were assessed for GLUT1, hexokinase-II (HKII), lactate dehydrogenase (LDHA), phosphorylated AKT (pAKT), and phosphorylated MAPK (pMAPK) at 0-72 h of treatment with 0.1 µM afatinib. Subcutaneous NCI-N87 tumor-bearing nude mice underwent [18F]FDG PET/MRI and HP-13C MRSI at baseline and 4 days after treatment with afatinib 10 mg/kg/day or vehicle (n = 10/group). Changes in PET and HP-13C MRSI metabolic parameters were compared between the two groups. Imaging findings were correlated with tumor growth and histopathology over 3 weeks of treatment. RESULTS: In vitro analysis showed a continuous decrease in LDHA, pAKT, and pMAPK in NCI-N87 compared to SNU16 cells within 72 h of treatment with afatinib, without a significant change in GLUT1 and HKII in either cell type. [18F]FDG PET of NCI-N87 tumors showed no significant change in PET measures at baseline and day 4 of treatment in either treatment group (SUVmean day 4/day 0: 2.7 ± 0.42/2.34 ± 0.38, p = 0.57 in the treated group vs. 1.73 ± 0.66/2.24 ± 0.43, p = 0.4 in the control group). HP-13C MRSI demonstrated significantly decreased lactate-to-pyruvate ratio (L/P) in treated tumors (L/P day 4/day 0: 0.83 ± 0.30/1.10 ± 0.20, p = 0.012 vs. 0.94 ± 0.20/0.98 ± 0.30, p = 0.75, in the treated vs. control group, respectively). Response to afatinib was confirmed with decreased tumor size over 3 weeks (11.10 ± 16.50 vs. 293.00 ± 79.30 mm3, p < 0.001, treated group vs. control group, respectively) and histopathologic evaluation. CONCLUSIONS: HP-13C MRSI is a more representative biomarker of early metabolic changes in response to pan-TKI in GCa than [18F]FDG PET and could be used for early prediction of response to targeted therapies.

Saliva is Comparable to Nasopharyngeal Swabs for Molecular Detection of SARS-CoV-2.

The continued need for molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the potential for self-collected saliva as an alternative to nasopharyngeal (NP) swabs for sample acquisition led us to compare saliva to NP swabs in an outpatient setting without restrictions to avoid food, drink, smoking, or tooth-brushing. A total of 385 pairs of NP and saliva specimens were obtained, the majority from individuals presenting for initial evaluation, and were tested on two high-sensitivity reverse transcriptase PCR (RT-PCR) platforms, the Abbott m2000 and Abbott Alinity m (both with limits of detection [LoD] of 100 copies of viral RNA/ml). Concordance between saliva and NP swabs was excellent overall (Cohen's κ = 0.93) for both initial and follow-up testing, for both platforms, and for specimens treated with guanidinium transport medium as preservative as well as for untreated saliva (κ = 0.88 to 0.95). Viral loads were on average 16× higher in NP specimens than saliva specimens, suggesting that only the relatively small fraction of outpatients (∼8% in this study) who present with very low viral loads (<1,600 copies/ml from NP swabs) would be missed by testing saliva instead of NP swabs when using sensitive testing platforms. Special attention was necessary to ensure leak-resistant specimen collection and transport. The advantages of self-collection of saliva, without behavioral restrictions, will likely outweigh a minor potential decrease in clinical sensitivity in individuals less likely to pose an infectious risk to others for many real-world scenarios, especially for initial testing. IMPORTANCE In general, the most accurate COVID-19 testing is hands-on and uncomfortable, requiring trained staff and a "brain-tickling" nasopharyngeal swab. Saliva would be much easier on both fronts, since patients could collect it themselves, and it is after all just spit. However, despite much interest, it remains unclear how well saliva performs in real-world settings when just using it in place of an NP swab without elaborate or cumbersome restrictions about not eating/drinking before testing, etc. Also, almost all studies of COVID-19 testing, whether of NP swabs, saliva, or otherwise, have been restricted to reporting results in the abstruse units of "CT values," which only mean something in the context of a specific assay and testing platform. Here, we compared saliva versus NP swabs in a real-world setting without restriction and report all results in natural units-the amount of virus being shed-showing that saliva is essentially just as good as NP swabs.

Visualizing the effects of lactate dehydrogenase (LDH) inhibition and LDH-A genetic ablation in breast and lung cancer with hyperpolarized pyruvate NMR.

In many tumors, cancer cells take up large quantities of glucose and metabolize it into lactate, even in the presence of sufficient oxygen to support oxidative metabolism. It has been hypothesized that this malignant metabolic phenotype supports cancer growth and metastasis, and that reversal of this so-called "Warburg effect" may selectively harm cancer cells. Conversion of glucose to lactate can be reduced by ablation or inhibition of lactate dehydrogenase (LDH), the enzyme responsible for conversion of pyruvate to lactate at the endpoint of glycolysis. Recently developed inhibitors of LDH provide new opportunities to investigate the role of this metabolic pathway in cancer. Here we show that magnetic resonance spectroscopic imaging of hyperpolarized pyruvate and its metabolites in models of breast and lung cancer reveal that inhibition of LDH was readily visualized through reduction in label exchange between pyruvate and lactate, while genetic ablation of the LDH-A isoform alone had smaller effects. During the acute phase of LDH inhibition in breast cancer, no discernible bicarbonate signal was observed and small signals from alanine were unchanged.

A microRNA/Runx1/Runx2 network regulates prostate tumor progression from onset to adenocarcinoma in TRAMP mice.

While decades of research have identified molecular pathways inducing and promoting stages of prostate cancer malignancy, studies addressing dynamic changes of cancer-related regulatory factors in a prostate tumor progression model are limited. Using the TRAMP mouse model of human prostate cancer, we address mechanisms of deregulation for the cancer-associated transcription factors, Runx1 and Runx2 by identifying microRNAs with reciprocal expression changes at six time points during 33 weeks of tumorigenesis. We molecularly define transition stages from PIN lesions to hyperplasia/neoplasia and progression to adenocarcinoma by temporal changes in expression of human prostate cancer markers, including the androgen receptor and tumor suppressors, Nkx3.1 and PTEN. Concomitant activation of PTEN, AR, and Runx factors occurs at early stages. At late stages, PTEN and AR are downregulated, while Runx1 and Runx2 remain elevated. Loss of Runx-targeting microRNAs, miR-23b-5p, miR-139-5p, miR-205-5p, miR-221-3p, miR-375-3p, miR-382-5p, and miR-384-5p, contribute to aberrant Runx expression in prostate tumors. Our studies reveal a Runx/miRNA interaction axis centered on PTEN-PI3K-AKT signaling. This regulatory network translates to mechanistic understanding of prostate tumorigenesis that can be developed for diagnosis and directed therapy.