High expression of circRNAs in muscle and stromal cell confound bulk tissue analyses in cancer research [NanoString]

Circular RNAs (circRNAs) are covalently closed molecules that are likely to play important roles in cancer development and progression. Hundreds of differentially expressed circRNAs between tumors and adjacent normal tissues have been identified in studies using RNA-sequencing or microarrays, emphasizing a strong translational potential. However, previous studies were performed using RNA from bulk tissues and the spatial expression patterns of most circRNAs are unknown. Here, we show that the majority of differentially expressed circRNAs from bulk tissue analyses of colon tumors relative to adjacent normal tissues are, surprisingly, not differentially expressed when comparing cancer cells directly with normal epithelial cells. By manipulating the proliferation rates of cells grown in culture, we find that these discrepancies are explained by circRNAs accumulating to high levels in quiescent muscle cells due to their high stability and, on the contrary, circRNAs are diluted to low levels in the fast-proliferating cancer cells due to their slow biogenesis rates. Thus, we observed striking changes in circRNA expression patterns within different sub-compartments of colon tumors and adjacent normal tissues. Likewise, we find that the high circRNA content in muscle cells can be a strong confounding factor in bulk analyses of circRNAs in bladder and prostate cancer. Together our findings emphasize the limitations of using bulk tissues for studying differential circRNA expression in cancer and highlight a particular need for spatial analysis in this field of research. This study included five stage II (TNM classification) colon cancer patients with tumors classified as T3 or T4. Paired normal tissues were also collected for all patients. Laser capture microdissection was performed for each of the five patient's tissues to separate two different sub-compartments from each cancer or normal tissue type. Cancer cells and tumor stroma were the two sub-compartments from the cancer tissues. Normal epithelium and muscularis propria were the two sub-compartments dissected from the adjacent normal tissues. RNA was extracted from the resulting samples. In addition, RNA was extracted from bulk cancer tissues and paired normal tissues. Finally, from the Caco-2 adenocarcinoma cell line, RNA was extracted and either treated with RNase R or mock-treated. In addition, RNA was extracted from three other colon adenocarcinoma cell lines. These samples were then interrogated for the 70 top high-abundance circRNA present in the colon. A list of housekeeping and tissue specific marker linear genes was also included in the panel.