Neuroblastoma (NBL) is a pediatric cancer that accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc occurs in 20% of NBL patients and is considered high risk as it correlates with aggressiveness, treatment resistance and poor prognosis. Even though the treatment strategies have improved in the recent years, the survival rate of high-risk NBL patients remain poor. Hence, it is crucial to explore new therapeutic avenues to sensitise NBL. Recently, bovine milk-derived extracellular vesicles (MEVs) have been proposed to contain anti-cancer properties. However, the impact of MEVs on NBL cells is not understood. In this study, we characterised MEVs using Western blotting, NTA and TEM. Importantly, treatment of NBL cells with MEVs decreased the proliferation and increased the sensitivity of NBL cells to doxorubicin. Temporal label-free quantitative proteomics of NBL cells highlighted the depletion of proteins involved in cell metabolism, cell growth and Wnt signalling upon treatment with MEVs. Furthermore, proteins implicated in cellular senescence and apoptosis were enriched in NBL cells treated with MEVs. For the first time, this study highlights the temporal proteomic profile that occurs in cancer cells upon MEVs treatment.

Apoptotic cells communicate to phagocytic cells through releasing soluble factors and apoptotic cell-derived extracellular vesicles. However, whether there are additional factors that remain attached at the site of cell death to signal to phagocytic cells is currently unknown. Here we show that apoptotic cell retraction generates a membrane-encased, F-actin-rich ‘footprint’ tightly anchored to the substrate that marks the site of cell death, coined ‘the FOotprint Of Death’ or FOOD. Formation of FOOD is observed frequently across many different cell types, apoptotic stimuli and surface composition. Mechanistically, FOOD formation is regulated by the protein kinase ROCK1. 3D time-lapse microscopy studies revealed that FOOD vesicularises into distinct large extracellular vesicles. These extracellular vesicles expose the ‘eat-me’ signal phosphatidylserine and can function to ‘flag’ the site of cell death to neighbouring phagocytes for efferocytosis. Under a viral infection setting, FOOD can harbour viral proteins and virions, and propagate infection to healthy cells. Together, this study has revealed another route of apoptotic cell-phagocyte communication.

Clinical management of cancer-associated cachexia, a multi-organ wasting syndrome, has been challenging without effective treatment strategies. An effective treatment that directly targets cancer-induced wasting is desperately needed to improve the quality of life and the survival of cancer patients. Recently, an antibiotic SFX was shown to have anti-tumour and anti-metastatic effects in mouse models of breast cancer. Hence, in this study, we examined the efficacy of SFX in the treatment of cancer-induced cachexia. C26 cachexic mice models were administered with SFX, and the tumour volume and body weight were regularly measured. Blood glucose, skeletal muscles, and adipose tissue were examined at the endpoint. Contrary to a previous study, SFX did not reduce the tumour volume in mice bearing C26 cells. Administration of SFX neither revealed any survival benefit nor rescued C26 cachectic mice from muscle wasting. Interestingly, SFX administration partially rescued (~10%) tumour-induced weight loss by preserving both the subcutaneous and intestinal fat mass. Together, these results suggest that the administration of SFX could partially rescue cancer-induced weight loss by inhibiting lipolysis. As anti-cachexia therapies are scarce, the results could facilitate the design of combinatorial therapies involving SFX, standard-of-care chemotherapeutics, and drugs that inhibit muscle atrophy for the treatment of cancer cachexia.

Proteases are enzymes that regulate substrates via proteolytic activation and coordinate essential cellular functions including DNA replication, DNA transcription, cell proliferation, differentiation, migration and apoptosis. However, techniques to identify proteolytic events in a high‐throughput manner is limited. PROtein TOpography and Migration Analysis Platform (PROTOMAP) is a technique that relies on mass spectrometry‐based proteomics to globally identify the shifts in the in‐gel migration of proteins and their corresponding fragments that are obtained by proteolysis. However, user‐friendly software tool to analyse the proteomic data to identify proteolytic events is needed. Here, we report Pep2Graph, a user‐friendly standalone tool that integrates peptide sequence information from in‐gel proteomics and presents the data as two‐dimensional peptographs with in‐gel migration, sequence coverage and MS/MS spectra counts. Pep2Graph (http://www.mathivananlab.org/Pep2Graph) allows users to utilize in‐gel proteomics data to study proteolytic events that may play a significant role in normal physiology and pathology.

Extracellular vesicles (EVs) are promising biomarkers, yet their proteomic analysis from plasma is hampered by low abundance and co-purification of contaminants (e.g., lipoproteins, platelets) and technical variability, particularly in small-volume animal models. We developed and validated a modular protocol integrating Size Exclusion Chromatography (SEC) with Strong Anion Exchange (SEC-SAX) specifically tailored for quantitative LC-MS proteomics from small starting volumes (150 μl of plasma). SEC alone successfully removed 99% of Albumin, and the SAX step significantly enriched EVs over contaminating lipoproteins. Downstream single pot solid phase enhanced (SP3) sample prep and STAGE tip solid phase extraction ensured maximum proteome depth. Critical confounding factors were objectively assessed: Platelet Factor 4 (PF4) was confirmed as a highly sensitive platelet marker, confirming the necessity of meticulous plasma preparation. Sample hemolysis impacted the plasma EV proteome data. As such, an objective measure (nanodrop spectrophotometer) of hemolysis and exclusion of hemolysed samples (heme >0.3 mg/ml) is recommended. The protocol is applicable to both human and mouse plasma as demonstrated by EV enrichment and quantification of biomarker proteins associated with neurodegenerative diseases from eight individual mouse plasma samples. Manuscript Highlights: Developmental workflow for a quantitative SEC-SAX protocol for EV proteomics from small plasma volumes (150 μl). A range of variables tested including SAX beads amount, digestion buffer, digestion time, STAGE tip solid phase extraction, SAX elution buffer and sample filtration. The SAX step significantly enhances EV proteome depth by increasing EV purity in relation to ApoB lipoproteins. Shows the impact of the major confounding factors of sample hemolysis and platelet contamination on the EV proteome. Platelet contamination increases the number and abundance of proteins detected including known disease biomarkers and sample hemolysis is associated with proteins derived from platelet and red blood cell derived EVs. Platelet Factor 4 (PF4) is identified and confirmed as a sensitive marker for platelet contamination. Applicable to both human and mouse plasma.

A 14-month-old Thoroughbred colt with hind limbs lameness was referred to Equine Hospital of Jeju National University. During physical examination, the patient could not walk in a straight line but wobbled especially with hind limbs. He hesitated when backing and crossed hind limbs while circling tightly by protracting the outer hind limb keeping the inner hind limb stationed. Stenosis was observed between the 2nd (C2) and 3rd (C3), and the C3 and 4th (C4) cervical vertebrae on radiographs. The colt diagnosed as cervical stenotic myelopathy (CSM) based on the clinical signs and radiological findings. To confirm the diagnosis, postmortem computed tomography (CT) and histopathologic examination were performed after the euthanasia. The CT images revealed severe compression of the spinal cavity and cord between C3 and C4 cervical vertebrae. Grossly, there was compression of the spinal cord between the C3 and C4 cervical vertebrae. Histopathologically, severe axonal swelling and demyelination characterized by vacuolation and cavity formation were observed in the white matter of the spinal cord, especially in C3 and C4. CSM is problematic in the horse racing industry because of abnormal walking. This case report of CSM may offer information for diagnosis of the disease in the equine veterinary fields in the Republic of Korea.