MicroRNAs (miRNAs) are deeply involved in tumor onset and progression, either behaving as tumor-promoting miRNAs (oncomiRNAs and metastamiRNAs) or as tumor suppressor miRNAs. This has a very important implication in diagnosis and/or prognosis, including the recent discovery that the pattern of circulating cell-free miRNAs in plasma allows us to perform molecular analyses on these non-invasive liquid biopsies. In order to develop in vivo experimental models for the validation of molecular assays aimed at detecting circulating miRNAs in plasma, we have inoculated nude mice with the HT-29, LS174T and LoVo human colorectal cancer cell lines. Xenotransplanted tumors were allowed to grow up to 0.5 cm3 in size. Blood was drawn at sacrifice, plasma was isolated by low speed centrifugation and was further treated to disrupt exosomes and denaturate miRNA-binding proteins using Qiazol solution. As internal control sequences, equal amount of C. elegans cel-miR-39 was spiked in all samples. Total microRNAs were purified with miRNeasy Serum/Plasma Kit (Qiagen). The levels of miR-141, miR-221 and miR-222 were assessed by Real-Time quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR). Mice xenotransplanted with HT29 cells displayed 2.3, 1.7 and 4.2 fold-increases in circulating miR-141, miR-221 and miR-222, respectively, compared to control mice. Mice transplanted with LoVo cells displayed increases of lesser magnitude (1.2, 1.7 and 2.2 for miR-141, miR-221 and miR-222), whereas mice transplanted with LS174T displayed a slight increase only for miR-222 (1.3 fold-increase). Circulating miRNAs levels were also correlated with their levels in cultured cells and in exosomes from cultured cells supernatants. Although the number of miRNAs taken into account is limited, these data clearly show, in a strictly controlled setting, that different colorectal carcinoma tumors give rise to very different liquid biopsy miRNA profiles. Therefore, wider panels of xenografts may be useful to recapitulate the clinical variety of colorectal tumors in human patients, estimate the minimal numbers of miRNAs in a diagnostic signature, and validate the overall applicative impact of oncomiRNAs in the liquid biopsy format.
A liquid biopsy model: Circulating miRNA levels in mice bearing colorectal carcinoma tumor xenografts
FINOTTI, Alessia;GASPARELLO, Jessica;FABBRI, Enrica;BIANCHI, Nicoletta;GAMBARI, Roberto
2016
Abstract
MicroRNAs (miRNAs) are deeply involved in tumor onset and progression, either behaving as tumor-promoting miRNAs (oncomiRNAs and metastamiRNAs) or as tumor suppressor miRNAs. This has a very important implication in diagnosis and/or prognosis, including the recent discovery that the pattern of circulating cell-free miRNAs in plasma allows us to perform molecular analyses on these non-invasive liquid biopsies. In order to develop in vivo experimental models for the validation of molecular assays aimed at detecting circulating miRNAs in plasma, we have inoculated nude mice with the HT-29, LS174T and LoVo human colorectal cancer cell lines. Xenotransplanted tumors were allowed to grow up to 0.5 cm3 in size. Blood was drawn at sacrifice, plasma was isolated by low speed centrifugation and was further treated to disrupt exosomes and denaturate miRNA-binding proteins using Qiazol solution. As internal control sequences, equal amount of C. elegans cel-miR-39 was spiked in all samples. Total microRNAs were purified with miRNeasy Serum/Plasma Kit (Qiagen). The levels of miR-141, miR-221 and miR-222 were assessed by Real-Time quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR). Mice xenotransplanted with HT29 cells displayed 2.3, 1.7 and 4.2 fold-increases in circulating miR-141, miR-221 and miR-222, respectively, compared to control mice. Mice transplanted with LoVo cells displayed increases of lesser magnitude (1.2, 1.7 and 2.2 for miR-141, miR-221 and miR-222), whereas mice transplanted with LS174T displayed a slight increase only for miR-222 (1.3 fold-increase). Circulating miRNAs levels were also correlated with their levels in cultured cells and in exosomes from cultured cells supernatants. Although the number of miRNAs taken into account is limited, these data clearly show, in a strictly controlled setting, that different colorectal carcinoma tumors give rise to very different liquid biopsy miRNA profiles. Therefore, wider panels of xenografts may be useful to recapitulate the clinical variety of colorectal tumors in human patients, estimate the minimal numbers of miRNAs in a diagnostic signature, and validate the overall applicative impact of oncomiRNAs in the liquid biopsy format.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
