Activated choline metabolism is a hallmark of carcinogenesis and tumor progression, which leads to elevated levels of phosphocholine and glycerophosphocholine in all types of cancer tested so far. Magnetic resonance spectroscopy applications have played a key role in detecting these elevated choline phospholipid metabolites. To date, the majority of cancer‐related studies have focused on phosphocholine and the Kennedy pathway, which constitutes the biosynthesis pathway for membrane phosphatidylcholine. Fewer and more recent studies have reported on the importance of glycerophosphocholine in cancer. In this review article, we summarize the recent literature on glycerophosphocholine metabolism with respect to its cancer biology and its detection by magnetic resonance spectroscopy applications.

BACKGROUND. Although aberrant glycosylation is recognized as a hallmark of cancer, glycosylation in clinical breast cancer (BC) metastasis has not yet been studied. While preclinical studies show that the glycocalyx coating of cancer cells is involved in adhesion, migration, and metastasis, glycosylation changes from primary tumor (PT) to various metastatic sites remain unknown in patients. METHODS. We investigated N-glycosylation profiles in 17 metastatic BC patients from our rapid autopsy program. Primary breast tumor, lymph node metastases, multiple systemic metastases, and various normal tissue cores from each patient were arranged on unique single-patient tissue microarrays (TMAs). We performed mass spectrometry imaging (MSI) combined with extensive pathology annotation of these TMAs, and this process enabled spatially differentiated cell-based analysis of N-glycosylation patterns in metastatic BC. RESULTS. N-glycan abundance increased during metastatic progression independently of BC subtype and treatment regimen, with high-mannose glycans most frequently elevated in BC metastases, followed by fucosylated and complex glycans. Bone metastasis, however, displayed increased core-fucosylation and decreased high-mannose glycans. Consistently, N-glycosylated proteins and N-glycan biosynthesis genes were differentially expressed during metastatic BC progression, with reduced expression of mannose-trimming enzymes and with elevated EpCAM, N-glycan branching, and sialyation enzymes in BC metastases versus PT. CONCLUSION. We show in patients that N-glycosylation of breast cancer cells undergoing metastasis occurs in a metastatic site–specific manner, supporting the clinical importance of high-mannose, fucosylated, and complex N-glycans as future diagnostic markers and therapeutic targets in metastatic BC. FUNDING. NIH grants R01CA213428, R01CA213492, R01CA264901, T32CA193145, Dutch Province Limburg “LINK”, European Union ERA-NET TRANSCAN2-643638.

Background Telomerase reverse transcriptase (TERT) is essential for tumor proliferation, including in low-grade oligodendrogliomas (LGOGs). Since TERT is silenced in normal cells, it is also a therapeutic target. Therefore, non-invasive methods of imaging TERT are needed. Here, we examined the link between TERT expression and metabolism in LGOGs, with the goal of leveraging this information for non-invasive magnetic resonance spectroscopy (MRS)-based metabolic imaging of LGOGs. Methods Immortalized normal human astrocytes with doxycycline-inducible TERT silencing, patient-derived LGOG cells, orthotopic tumors and LGOG patient biopsies were studied to determine the mechanistic link between TERT expression and glucose metabolism. The ability of hyperpolarized [U- 13C, U- 2H]-glucose to non-invasively assess TERT expression was tested in live cells and orthotopic tumors. Results TERT expression was associated with elevated glucose flux through the pentose phosphate pathway (PPP), elevated NADPH, which is a major product of the PPP, and elevated GSH, which is maintained in a reduced state by NADPH. Importantly, hyperpolarized [U- 13C, U- 2H]-glucose metabolism via the PPP non-invasively reported on TERT expression and response to TERT inhibition in patient-derived LGOG cells and orthotopic tumors. Mechanistically, TERT acted via the sirtuin SIRT2 to upregulate the glucose transporter GLUT1 and the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase. Conclusions We have, for the first time, leveraged a mechanistic understanding of TERT-associated metabolic reprogramming for non-invasive imaging of LGOGs using hyperpolarized [U- 13C, U- 2H]-glucose. Our findings provide a novel way of imaging a hallmark of tumor immortality and have the potential to improve diagnosis and treatment response assessment for LGOG patients.

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ADP-ribosylation is a protein modification responsible for biological processes such as DNA repair, RNA regulation, cell cycle and biomolecular condensate formation. Dysregulation of ADP-ribosylation is implicated in cancer, neurodegeneration and viral infection. We developed ADPriboDB (adpribodb.leunglab.org) to facilitate studies in uncovering insights into the mechanisms and biological significance of ADP-ribosylation. ADPriboDB 2.0 serves as a one-stop repository comprising 48 346 entries and 9097 ADP-ribosylated proteins, of which 6708 were newly identified since the original database release. In this updated version, we provide information regarding the sites of ADP-ribosylation in 32 946 entries. The wealth of information allows us to interrogate existing databases or newly available data. For example, we found that ADP-ribosylated substrates are significantly associated with the recently identified human protein interaction networks associated with SARS-CoV-2, which encodes a conserved protein domain called macrodomain that binds and removes ADP-ribosylation. In addition, we create a new interactive tool to visualize the local context of ADP-ribosylation, such as structural and functional features as well as other post-translational modifications (e.g. phosphorylation, methylation and ubiquitination). This information provides opportunities to explore the biology of ADP-ribosylation and generate new hypotheses for experimental testing.

ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. We herein show that an adenosine analogue with a terminal alkyne functionality at position 2 of the adenine (2-alkyne adenosine or 2YnAd) is suitable for selective enrichment, fluorescence detection and mass spectrometry proteomics analysis of the candidate ADP-ribosylome in mammalian cells. Although similar labelling profiles were observed via fluorescence imaging for 2YnAd and 6YnAd, a previously reported clickable NAD + precursor, quantitative mass spectrometry analysis of the two probes in MDA-MB-231 breast cancer cells revealed a significant increase in protein coverage of the 2YnAd probe. To facilitate global enrichment of ADP-ribosylated proteins, we developed a dual metabolic labelling approach that involves simultaneous treatment of live cells with both 2YnAd and 6YnAd. By combining this dual metabolic labelling strategy with highly sensitive tandem mass tag (TMT) isobaric mass spectrometry and hierarchical Bayesian analysis, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib.

Identification and classification of leukemia cells in a rapid and label-free fashion is clinically challenging and thus presents a prime arena for implementing new diagnostic tools. Quantitative phase imaging, which maps optical path length delays introduced by the specimen, has been demonstrated to discern cellular phenotypes based on differential morphological attributes. Rapid acquisition capability and the availability of label-free images with high information content have enabled researchers to use machine learning (ML) to reveal latent features. We developed a set of ML classifiers, including convolutional neural networks, to discern healthy B cells from lymphoblasts and classify stages of B cell acute lymphoblastic leukemia. Here, we show that the average dry mass and volume of normal B cells are lower than those of cancerous cells and that these morphologic parameters increase further alongside disease progression. We find that the relaxed training requirements of a ML approach are conducive to the classification of cell type, with minimal space, training time, and memory requirements. Our findings pave the way for a larger study on clinical samples of acute lymphoblastic leukemia, with the overarching goal of its broader use in hematopathology, where the prospect of objective diagnoses with minimal sample preparation remains highly desirable.

One‐third of patients discharged from hospitals to skilled nursing facilities (SNF) are sent back to the Emergency Department (ED) within 30 days. Little is known about those patients who are discharged from the ED directly back to SNF. We considered these ED visits as potentially avoidable since they did not result in observation or hospitalization stay. Using a retrospective chart review of 1010 patients with ED visits within 14‐days of discharge to SNF from University of Pennsylvania health system (UPHS) in 2020–2021, we identified 202 patients with potentially avoidable ED visits among medical and surgical patients. The most common reasons for these ED visits were mechanical falls (17.3%), postoperative problems (16.8%), and cardiac or pulmonary complaints (11.4%). Future interventions to decrease avoidable ED visits from SNFs should aim to provide access for SNF patients to receive timely outpatient lab and imaging services and postoperative follow‐ups.