Analysis of microRNAs in Urinary Exosomes of Tumor‐bearing Mice as a Non‐invasive Tool for Lymphoma Diagnosis

Abstract

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.