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Page 6 of 11 Gasparello et al. J Cancer Metastasis Treat 2019;5:52 I http://dx.doi.org/10.20517/2394-4722.2019.17

cells was found the most efficient for miRNA detection. The HT-29 cells were derived from a KRAS-WT,
differentiated colorectal adenocarcinoma [72,73] , while LoVo cells (originally described as Dukes’ type C, grade
IV) harbor a heterozygous KRAS c.38G>A mutation (G13D) .
[74]

TUMOR XENOGRAFTS AND PLASMA PREPARATION
Figure 1B shows the study workflow based on the tumor xenografts as models for liquid biopsy to assess
plasma levels of circulating miRNAs. In this study workflow, miRNAs are compared considering (1) in vitro
cultured tumor cells; (2) tumor xenografts; and (3) blood plasma samples. The HT-29 and LoVo cell lines were
selected as proxies of clinically evident cancers and sources of soluble analytes. MicroRNAs were extracted
from both cell lines, matched tumor tissue and blood plasma samples and were then subjected to ddPCR
and RT-qPCR analysis. Tumor xenotransplants were established by inoculating HT-29 and LoVo cells in the
flank of 4-month old Nu/CD1 mice. Tumors were taken at sacrifice along with blood. Frozen tissues were
used as the source of miRNAs. For the analysis of ctmiRNA, blood plasma was treated to disrupt exosomes
and denature miRNA-binding proteins with QIAzol Lysis Reagent. After the addition of 400 amoles of cel-
miR-39-3p (an equalizer), total RNA was purified and reverse transcribed. Finally, droplet digital PCR (RT-
ddPCR) assays for microRNA content analysis were performed to quantify the levels of miR-221-3p [37,75] and
miR-222-3p . Droplets were analyzed using the QX200 Droplet Reader, and data analysis was performed
[76]
with QuantaSoft version 1.7.4 (Bio-Rad, Hercules, CA, USA).

TUMOR XENOGRAFTS AND PLASMA PREPARATION: MAJOR RESULTS
The main point of this study is focused on determining whether the pattern of plasma miRNA content
recapitulates HT-29 and LoVo cells and xenografted tumors. A representative example of miR-222-3p
content is shown in Figure 2A and all the quantitative data for miR-221-3p and miR-222-3p are presented in
Figure 2B. The miRNA levels were independently assessed by RT-qPCR and ddPCR results, obtaining similar
results, as reported elsewhere . Of course, in the quantitative analysis shown in Figure 2B and concerning
[55]
the plasma miRNA quantitation, we have taken into account the fact that cross-species miRNA homology
might influence our in vivo results. Accordingly, we quantitated baseline, endogenous miR-221-3p and
miR-222-3p levels in tumor-free, healthy nude mice. As expected (the sequences of mouse miR-221-3p and
miR-222-3p are identical to those found in human cells) both RT-ddPCR and RT-qPCR demonstrated that
circulating miRNAs were detectable even in the absence of tumor growth. However, the differences between
tumor-bearing and tumor-free mice were clearly appreciable for both miRNAs. Figure 2B shows that the
miR-222-3p content is higher than miR-221-3p content in HT-29 and LoVo cells, and in tumor and plasma
samples isolated from HT-29 and LoVo xenografted mice, despite the miR-222/miR-221 ratio is much higher
in plasma in comparison with cell and tumors. This is consistent with the “gateway” effect mentioned above
and discussed in deep in Gasparello et al . This issue is particularly of interest, since detailed knowledge of
[55]
the molecular mechanisms underlying the release of circulating analytes is still lacking. Alternatively, the in
vivo response of xenotransplanted mice to tumor cell injection might contribute to the reported unbalanced
content of miR-222/miR-221. The proposed system is expected to help in verifying the underlying cellular
mechanisms.

CONCLUSION
Circulating miRNAs have been recently used as biological markers for early diagnosis, prognosis, prediction
of response to therapy and clinical outcome, particularly in a liquid biopsy setting [1-11,77-79] . Liquid biopsy is
a powerful tool applicable to all or most human cancers, including colorectal, lung, melanoma, and breast
neoplasms [80,81] . From a more general viewpoint, tumor-xenotransplanted mice and other in vivo models may
have an important role because they resolve biological variables from technical variables (such as handling
and storage of biological fluids, pre-analytical processing, as well as DNA and RNA isolation protocols) that