13C-SpaceM: Spatial single-cell isotope tracing reveals heterogeneity of de novo fatty acid synthesis in cancer.

Metabolism has emerged as a key factor in homeostasis and disease including cancer. Yet, little is known about the heterogeneity of metabolic activity of cancer cells due to the lack of tools to directly probe it. Here, we present a novel method, 13C-SpaceM for spatial single-cell isotope tracing of glucose-dependent de novo lipogenesis. The method combines imaging mass spectrometry for spatially-resolved detection of 13C6-glucose-derived 13C label incorporated into esterified fatty acids with microscopy and computational methods for data integration and analysis. We validated 13C-SpaceM on a spatially-heterogeneous normoxia-hypoxia model of liver cancer cells. Investigating cultured cells, we revealed single-cell heterogeneity of lipogenic acetyl-CoA pool labelling degree upon ACLY knockdown that is hidden in the bulk analysis and its effect on synthesis of individual fatty acids. Next, we adapted 13C-SpaceM to analyze tissue sections of mice harboring isocitrate dehydrogenase (IDH)-mutant gliomas. We found a strong induction of de novo fatty acid synthesis in the tumor tissue compared to the surrounding brain. Comparison of fatty acid isotopologue patterns revealed elevated uptake of mono-unsaturated and essential fatty acids in the tumor. Furthermore, our analysis uncovered substantial spatial heterogeneity in the labelling of the lipogenic acetyl-CoA pool indicative of metabolic reprogramming during microenvironmental adaptation. Overall, 13C-SpaceM enables novel ways for spatial probing of metabolic activity at the single cell level. Additionally, this methodology provides unprecedented insight into fatty acid uptake, synthesis and modification in normal and cancerous tissues.

Investigating post-traumatic syringomyelia and local fluid osmoregulation via a rat model.

BACKGROUND: Syringomyelia (SM) is characterized by the development of fluid-filled cavities, referred to as syrinxes, within the spinal cord tissue. The molecular etiology of SM post-spinal cord injury (SCI) is not well understood and only invasive surgical based treatments are available to treat SM clinically. This study builds upon our previous omics studies and in vitro cellular investigations to further understand local fluid osmoregulation in post-traumatic SM (PTSM) to highlight important pathways for future molecular interventions. METHODS: A rat PTSM model consisting of a laminectomy at the C7 to T1 level followed by a parenchymal injection of 2 µL quisqualic acid (QA) and an injection of 5 µL kaolin in the subarachnoid space was utilized 6 weeks after initial surgery, parenchymal fluid and cerebrospinal fluid (CSF) were collected, and the osmolality of fluids were analyzed. Immunohistochemistry (IHC), metabolomics analysis using LC-MS, and mass spectrometry-based imaging (MSI) were performed on injured and laminectomy-only control spinal cords. RESULTS: We demonstrated that the osmolality of the local parenchymal fluid encompassing syrinxes was higher compared to control spinal cords after laminectomy, indicating a local osmotic imbalance due to SM injury. Moreover, we also found that parenchymal fluid is more hypertonic than CSF, indicating establishment of a local osmotic gradient in the PTSM injured spinal cord (syrinx site) forcing fluid into the spinal cord parenchyma to form and/or expand syrinxes. IHC results demonstrated upregulation of betaine, ions, water channels/transporters, and enzymes (BGT1, AQP1, AQP4, CHDH) at the syrinx site as compared to caudal and rostral sites to the injury, implying extensive local osmoregulation activities at the syrinx site. Further, metabolomics analysis corroborated alterations in osmolality at the syrinx site by upregulation of small molecule osmolytes including betaine, carnitine, glycerophosphocholine, arginine, creatine, guanidinoacetate, and spermidine. CONCLUSIONS: In summary, PTSM results in local osmotic disturbance that propagates at 6 weeks following initial injury. This coincides with and may contribute to syrinx formation/expansion.

Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.