Muscle stem cells (MuSCs) hold great potential as a regenerative therapeutic but have met numerous challenges in treating systemic muscle diseases. Muscle stem cell-derived extracellular vesicles (MuSC-EVs) may overcome these limitations. We assessed the number and size distribution of extracellular vesicles (EVs) released by MuSCs ex vivo, determined the extent to which MuSC-EVs deliver molecular cargo to myotubes in vitro, and quantified MuSC-EV-mediated restoration of mitochondrial function following oxidative injury. MuSCs released an abundance of EVs in culture. MuSC-EVs delivered protein cargo into myotubes within 2 h of incubation. Fluorescent labeling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. Oxidatively injured myotubes demonstrated a significant decline in maximal oxygen consumption rate and spare respiratory capacity relative to untreated myotubes. Remarkably, subsequent treatment with MuSC-EVs significantly improved maximal oxygen consumption rate and spare respiratory capacity relative to the myotubes that were damaged but received no subsequent treatment. Surprisingly, MuSC-EVs did not affect mitochondrial function in undamaged myotubes, suggesting the cargo delivered is able to repair but does not expand the existing mitochondrial network. These data demonstrate that MuSC-EVs rapidly deliver proteins into myotubes, a portion of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged myotubes.

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3 ABSTRACTSatellite cells (SCs) are muscle-specific stem cells that have a central role in muscle remodeling. Despite their therapeutic potential, SC-based therapies have been met with numerous logistical challenges, limiting their ability to effectively treat systemic muscle diseases, such as Duchenne muscular dystrophy (DMD). Delivery of SC-derived extracellular vesicles (SC-EVs) may unlock the potential offered by SCs and overcome their numerous limitations. Purpose: The purpose of this investigation was to determine the extent to which SC-EVs could restore mitochondrial function in cultured myotubes following oxidative injury. Methods: SC-EVs were isolated from cultured SCs from C57 mice and quantified using nanoparticle tracking analysis (NTA). C2C12 myotubes were cultured and divided into four treatment groups: untreated control, treated for 24 h with SC-EV, 24 h exposure to 50 μ M H 2 O 2 followed by a 24 h recovery period with no treatment, or 24 h exposure to 50 μ M H 2 O 2 followed by a 24 h treatment with SC-EV. Inter-group differences in mitochondrial function were assessed via one-way ANOVA with Tukey post hoc analysis (p<0.05). Results: Given the seeding density used, we calculated that each SC releases approximately 2.35 x 10 5 ± 3.10 x 10 4 EVs per 24 h. Further, using fluorescent microscopy, we verified SC-EVs deliver cargo into myotubes, some of which was localized to the mitochondria. H 2 O 2 exposure resulted in a 42% decline in peak mitochondrial respiration (p=0.0243) as well as a 46% reduction in spare respiratory capacity (p=0.0185) relative to the untreated control group. Subsequent treatment with SC-EVs (3.12x10 8 SC-EV; 24 h) following H 2 O 2 exposure restored 76% of peak mitochondrial respiration (p=0.0187) and 84% of spare respiratory capacity in the damaged myotubes (p=0.0198). SC-EVs did not affect mitochondrial function in the undamaged myotubes. Conclusion: Collectively, these data demonstrate SC-EVs may represent a novel therapeutic approach for treatment of myopathies associated with mitochondrial dysfunction.

Lymphoma consists of a subgroup of immunological cancers, which arise from malignant B and T lymphocytes. Comprising approximately 4% of cancers in the United States, lymphoma accounts for up to 19,940 deaths per year. Surgical biopsy is the primary method for lymphoma staging and diagnosis. Unfortunately, there are many risks associated with this procedure, including cosmetic disfigurement, seeding of malignant cells into previously unaffected tissue, and expense incurred by patients. Identification of unique cancer‐related biomarkers in urinary exosomes may provide a novel non‐invasive and cost‐effective tool for lymphoma diagnosis. Although analyses of biomarkers obtained from urinary exosomes have been used to evaluate urological cancers, these methods have not yet been translated to non‐urological pathologies, such as lymphomas. The objective for this study was to analyze the profile of microRNAs (miRNAs) obtained from urinary exosomes of mice bearing lymphoma tumors and compare it to miRNAs identified in urinary exosomes of tumor free (‐) control mice. Male and female C57BL/6 mice were injected with either 2.5x105 mouse EL‐4 lymphoma cells [tumor (+) mice, n=6] or phosphate‐buffered saline [tumor (‐) mice, n=6]. Tumor growth was monitored for up to 20 days with collection of serum, tumor tissues, and organs at the end of this period. Urine was collected for 48 hours beginning on day 17. Extraction of urinary exosomes was followed by total RNA isolation and RT‐qPCR using a set of PCR arrays consisting of 709 mouse‐specific miRNA primers. Fold changes in miRNA expression were quantified using the ∆∆Ct method. Mice developed tumors by day 13 with initial tumor appearance around day seven. There were no statistically significant differences between final tumor mass, body weights, or food and water intake in tumor (+) versus tumor (‐) mice. RT‐qPCR arrays of miRNAs extracted from urinary exosomes revealed 464 differentially expressed miRNAs between tumor (+) and tumor (‐) mice. Specifically, the expression of two miRNAs, significantly increased in urinary exosomes of tumor (+) mice compared to tumor (‐) mice, was also increased in the lymphoma tissue of origin. One miRNA with a significantly decreased expression in the urinary exosomes of tumor (+) mice was found to have a decreased expression in the lymphoma tissue of origin. Expression of miRNAs in mouse tissue was analyzed by RT‐qPCR. These results revealed that mice challenged with lymphoma tumors are releasing tumor‐specific miRNAs in their urine. These findings are in support of future clinical studies on the use of miRNA analysis in urinary exosomes for non‐invasive diagnostics of lymphoma patients.

Lymphoma accounts for approximately 4% of cancers in the United States, with an estimated 20,910 number of deaths per year. The standard method of diagnosis and staging of lymphoma involves surgical biopsy of the tumor - a procedure that has many negative associated risks. Exosomes are extracellular vesicles secreted in biological fluids that can serve as “liquid biomarkers”. Identification of unique cancer-related biomarkers in urinary exosomes may provide a novel non-invasive and cost-effective tool for lymphoma diagnosis. Analyses of biomarkers obtained from urinary exosomes have been used to evaluate urological cancers, however, these methods have not yet been translated to non-urological pathologies, such as lymphomas. The objective for this study was to determine the profile of microRNAs (miRNAs) expressed in urinary exosomes of mice challenged with lymphoma and compare it to miRNAs identified in urinary exosomes of control mice. Male and female C57BL/6 mice, (tumor (+), n=12), were injected with either 2.5x105 mouse EL-4 lymphoma cells or phosphate-buffered saline (tumor (-), n=12). Tumor growth was monitored for 20 days. Urine was collected for 48 hours starting on day 17 and serum, tumor tissues, and organs were collected on day 20. Extraction of urinary exosomes was followed by total RNA isolation and RT-qPCR for which a set of PCR arrays consisting of 709 mouse-specific miRNA primers was used. Fold changes in miRNA expression were quantified using the ΔΔCt method. Mice developed tumors by day 13 with initial tumor appearance around day seven. There were no statistically significant differences between final tumor mass, body weight, or food and water intake in tumor (+) versus tumor (-) mice. RT-qPCR arrays of miRNAs extracted from urinary exosomes revealed 464 miRNAs that were differentially expressed between tumor (+) and tumor (-). 215 miRNAs were up-regulated, while 249 miRNAs were down-regulated in tumor (+) mice. These results will be compared to miRNAs from serum and tumor tissues to identify tumor-specific miRNAs that can be used for potential application in the clinical setting. Citation Format: Brittany Wilson, Rebekah Betar, Alexander Martin, Zackaria Niazi, Michael Boyer, Lori Winter, Victor Babich, Francesca Di Sole, Elitsa Ananieva. microRNA expression profile in urinary exosomes is dependent on non-invasive lymphoma induction in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2377.