Extracellular vesicle-based liquid biopsy biomarkers and their application in precision immuno-oncology.

While the field of precision oncology is rapidly expanding and more targeted options are revolutionizing cancer treatment paradigms, therapeutic resistance particularly to immunotherapy remains a pressing challenge. This can be largely attributed to the dynamic tumor-stroma interactions that continuously alter the microenvironment. While to date most advancements have been made through examining the clinical utility of tissue-based biomarkers, their invasive nature and lack of a holistic representation of the evolving disease in a real-time manner could result in suboptimal treatment decisions. Thus, using minimally-invasive approaches to identify biomarkers that predict and monitor treatment response as well as alert to the emergence of recurrences is of a critical need. Currently, research efforts are shifting towards developing liquid biopsy-based biomarkers obtained from patients over the course of disease. Liquid biopsy represents a unique opportunity to monitor intercellular communication within the tumor microenvironment which could occur through the exchange of extracellular vesicles (EVs). EVs are lipid bilayer membrane nanoscale vesicles which transfer a plethora of biomolecules that mediate intercellular crosstalk, shape the tumor microenvironment, and modify drug response. The capture of EVs using innovative approaches, such as microfluidics, magnetic beads, and aptamers, allow their analysis via high throughput multi-omics techniques and facilitate their use for biomarker discovery. Artificial intelligence, using machine and deep learning algorithms, is advancing multi-omics analyses to uncover candidate biomarkers and predictive signatures that are key for translation into clinical trials. With the increasing recognition of the role of EVs in mediating immune evasion and as a valuable biomarker source, these real-time snapshots of cellular communication are promising to become an important tool in the field of precision oncology and spur the recognition of strategies to block resistance to immunotherapy. In this review, we discuss the emerging role of EVs in biomarker research describing current advances in their isolation and analysis techniques as well as their function as mediators in the tumor microenvironment. We also highlight recent lung cancer and melanoma studies that point towards their application as predictive biomarkers for immunotherapy and their potential clinical use in precision immuno-oncology.

Comparative assessment of hepatoprotective properties of Artesunate and flavonoids from Artemisia annua on acetaminophen and carbon tetrachloride-induced cytotoxicity in primary mice hepatocytes.

Background: Artesunate (ART) is a semi-synthetized molecule from Artemisinin, an active compound isolated from the medicinal plant Artemisia annua, widely used for the treatment of malaria. Previous studies reported that ART may exert a dual effect on the liver. Accordingly, this study investigated the potential protective action of ART against Acetaminophen (APAP) and Carbon tetrachloride (CCl4)-induced hepatotoxicity in primary mice hepatocytes, in comparison to that of flavonoid extracted from A. annua (FAA). In addition, the antioxidant properties of FAA were also assessed. Methods: The antioxidant activities of FAA and Ascorbic acid (ASC) (0.01-100 µg/mL) were assessed through inhibition of lipid peroxidation, reduction of ferric and phosphomolydenum, and hydroxyl and DPPH radicals scavenging assays. The hepatoprotective effects of FAA and ART (0.1-100 µg/mL) were evaluated against APAP (11 mM) or CCl4 (4 mM) induced oxidative damage in primary mouse hepatocytes. Biochemical parameters associated with hepatotoxicity assessed include cell viability, cell membrane integrity, cellular glutathione, and antioxidant enzyme activities. Results: The obtained finding revealed FAA displayed a remarkable antioxidant activities as evidenced by the low IC50/EC50 values (3.85-19.32 µg/mL), comparable to that of ASC (3.26-18.04 µg/mL). When tested at 10 µg/mL, both FAA and ART significantly (p˂0.05) preserved cell viability, inhibited alanine aminotransferase leakage and lipid membrane peroxidation, and restored superoxide dismutase and catalase activities and glutathione content induced by APAP or CCl4 in a similar way as Silymarin. However, ART showed a significant (p˂0.05) cytotoxic effect on hepatocytes at 100 and 1000 µg/mL and did not confer obvious protection at 100 µg/mL. Conclusion: Overall, our data demonstrated that ART harms mice hepatocytes at high concentration while conferring relative protection against APAP and CCl4-hepatotoxicity at low concentration. In contrast, FAA effectively protects liver cells without cytotoxicity effect, event at 100 µg/mL. Accordingly, ART should be given to the patient only under a medical prescription.

Evaluating the mitochondrial activity and inflammatory state of dimethyl sulfoxide differentiated PLB-985 cells.

Neutrophils play a key role in the innate immunity with their ability to generate and release inflammatory mediators that promote the inflammatory response and consequently restore the hemostasis. As active participants in several steps of the normal inflammatory response, neutrophils are also involved in chronic inflammatory diseases such as asthma, atherosclerosis, and arthritis. Given their dual role in the modulation of inflammation, regulating the inflammatory response of neutrophils has been suggested as an important therapeutic approach by numerous researchers. The neutrophils have a relatively short lifespan, which can be problematic for some in vitro experiments. To address this issue, researchers have used the human monomyelocyte cell line PLB-985 as an in vitro model for exploratory experiments addressing neutrophil-related physiological functions. PLB-985 cells can be differentiated into a neutrophil-like phenotype upon exposure to several agonists, including dimethyl sulfoxide (DMSO). Whether this differentiation of PLB-985 affects important features related to the neutrophil's normal functions (i.e., mitochondrial activity, eicosanoid production) remains elusive, and characterizing these changes will be the focal point of this study. Our results indicate that the differentiation affected the proliferation of PLB-985 cells, without inducing apoptosis. A significant decrease in mitochondrial respiration was observed in differentiated PLB-985 cells. However, the overall mitochondria content was not affected. Immunoblotting with mitochondrial antibodies revealed a strong modulation of the succinate dehydrogenase A, superoxide dismutase 2, ubiquinol-cytochrome c reductase core protein 2 and ATP synthase subunit α in differentiated PLB-985 cells. Finally, eicosanoids (leukotriene B4, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids) production was significantly increased in differentiated cells. In summary, our data demonstrate that the differentiation process of PLB-985 cells does not impact their viability despite a reduced respiratory state of the cells. This process is also accompanied by modulation of the inflammatory state of the cell. Of importance, our data suggest that PLB-985 cells could be suitable in vitro candidates to study mitochondrial-related dysfunctions in inflammatory diseases.

Multi-omics Reveal that c-Src Modulates the Mitochondrial Phosphotyrosine Proteome and Metabolism According to Nutrient Availability.

BACKGROUND/AIMS: Src kinase family members, including c-Src, are involved in numerous signaling pathways and have been observed inside different cellular compartments. Notably, c-Src modulates carbohydrate and fatty acid metabolism and is involved in the metabolic rewiring of cancer cells. This kinase is found within mitochondria where it targets different proteins to impact on the organelle functions and overall metabolism. Surprisingly, no global metabolic characterization of Src has been performed although c-Src knock-out mice have been available for 30 years. Considering that c-Src is sensitive to various metabolites, c-Src might represent a crucial player in metabolic adjustments induced by nutrient stress. The aim of this work was to characterize the impact of c-Src on mitochondrial activity and overall metabolism using multi-omic characterization. METHODS: Src+/+ and Src-/- mice were fed ad libitum or fasted during 24h and were then analyzed using multi-omics. RESULTS: We observed that deletion of c-Src is linked to lower phosphorylation of Y412-NDUFA8, inhibition of oxygen consumption and accumulation of metabolites involved in glycolysis, TCA cycle and amino acid metabolism in mice fed ad libitum. Finally, metabolomics and (phosphotyrosine) proteomics are differently impacted by Src according to nutrient availability. CONCLUSION: The findings presented here highlight that c-Src reduces mitochondrial metabolism and impacts the metabolic adjustment induced by nutrient stress.

Intramitochondrial Src kinase links mitochondrial dysfunctions and aggressiveness of breast cancer cells.

High levels and activity of Src kinase are common among breast cancer subtypes, and several inhibitors of the kinase are currently tested in clinical trials. Alterations in mitochondrial activity is also observed among the different types of breast cancer. Src kinase is localized in several subcellular compartments, including mitochondria where it targets several proteins to modulate the activity of the organelle. Although the subcellular localization of other oncogenes modulates the potency of known treatments, nothing is known about the specific role of intra-mitochondrial Src (mtSrc) in breast cancer. The aim of this work was to determine whether mtSrc kinase has specific impact on breast cancer cells. We first observed that activity of mtSrc is higher in breast cancer cells of the triple negative subtype. Over-expression of Src specifically targeted to mitochondria reduced mtDNA levels, mitochondrial membrane potential and cellular respiration. These alterations of mitochondrial functions led to lower cellular viability, shorter cell cycle and increased invasive capacity. Proteomic analyses revealed that mtSrc targets the mitochondrial single-stranded DNA-binding protein, a regulator of mtDNA replication. Our findings suggest that mtSrc promotes aggressiveness of breast cancer cells via phosphorylation of mitochondrial single-stranded DNA-binding protein leading to reduced mtDNA levels and mitochondrial activity. This study highlights the importance of considering the subcellular localization of Src kinase in the development of potent therapy for breast cancer.

Linking Mitochondria and Synaptic Transmission: The CB1 Receptor.

CB1 receptors are functionally present within brain mitochondria (mtCB1), although they are usually considered specifically targeted to plasma membrane. Acute activation of mtCB1 alters mitochondrial ATP generation, synaptic transmission, and memory performance. However, the detailed mechanism linking disrupted mitochondrial metabolism and synaptic transmission is still uncharacterized. CB1 receptors are among the most abundant G protein-coupled receptors in the brain and impact on several processes, including fear coping, anxiety, stress, learning, and memory. Mitochondria perform several key physiological processes for neuronal homeostasis, including production of ATP and reactive oxygen species, calcium buffering, metabolism of neurotransmitters, and apoptosis. It is therefore possible that acute activation of mtCB1 impacts on these different mitochondrial functions to modulate synaptic transmission. In reviewing and integrating across the literature in this area, we describe the possible mechanisms involved in the regulation of brain physiology by mtCB1 receptors.