Liquid biopsy in mice bearing colorectal carcinoma xenografts: Gateways regulating the levels of circulating tumor DNA (ctDNA) and miRNA (ctmiRNA)

Background: Circulating tumor DNA (ctDNA) and miRNA (ctmiRNA) are promising biomarkers for early tumor diagnosis, prognosis and monitoring, and to predict therapeutic response. However, a clear understanding of the fine control on their circulating levels is still lacking. Methods: Three human colorectal carcinoma cell lines were grown in culture and as tumor xenograft models in nude mice. Chip-based and droplet digital PCR platforms were used to systematically and quantitatively assess the levels of DNAs and miRNAs released into the culture supernatants and mouse blood plasma. Results: Strikingly, mutated DNAs from the same (KRAS) and different (PIK3CA and FBWX7) genomic loci were differentially detected in culture supernatants and blood, with LS174T releasing 25 to 60 times less DNA in culture, but giving rise to 7 to 8 times more DNA in blood than LoVo cells. Greater LS174T ctDNA accumulation occurred in spite of similar CD31 immunostaining (micro-vascularization) and lesser proliferation and tissue necrosis as compared to LoVo. As to the three selected miRNAs (miR-221, miR-222 and miR-141), all of them were constitutively present in the plasma of tumor-free mice. Micro-RNA miR-141 was released into HT-29 cell supernatants 10 and 6.5 times less abundantly with respect to LoVo and LS174T, respectively; on the contrary, release of miR-141 in blood of HT-29 xenografted mice was found similar to that observed in LoVo and LS174T mice. Conclusions: Taken together, our results support the existence of multiple, finely tuned (non-housekeeping) control gateways that selectively regulate the release/accumulation of distinct ctDNA and miRNA species in culture and tumor xenograft models. Different xenografts (proxies of different patients) considerably differ in gateway usage, adding several layers of complexity to the well-known idea of molecular heterogeneity. We predict that even high tissue representation of mutated DNA and miRNA may result in insufficient diagnostic analyte representation in blood. In this respect, our data show that careful modeling in mice may considerably help to alleviate complexity, for instance by pre-screening for the most abundant circulating analytes in enlarged sets of tumor xenografts.