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Page 4 of 11 Gasparello et al. J Cancer Metastasis Treat 2019;5:52 I http://dx.doi.org/10.20517/2394-4722.2019.17

detection techniques have been proposed in recent years such as bead-based flow cytometry but at the
[60]
moment they are employed only for miRNA detection in tissues or cellular samples.

EXPERIMENTAL MODEL SYSTEMS FOR LIQUID BIOPSY
Given the fast pace of technological evolution and the clinical complexity of human cancers, there is an
increasing need for experimental in vivo systems and associated validation technologies. A robust analysis
of bio-fluids must acknowledge the fact that liquid biopsy is a complex strategy requiring the achievements
of several key points, including pre-analytical steps, post-analytical optimization, and careful selection of
optimal analytes for specific biological queries. In vivo model systems might be very useful to address and
isolate these many individual variables (that are both technical and biological), and validate complex multi-
step approaches. It is surprising, in this respect, that only few reports are available focusing on the use of
animal models. In vivo model systems for the detection of circulating tumor cells and DNA have been
proposed [61-63] . These include injection of cancer cells into their orthotopic site of origin (e.g., a “recap” of
natural tumor onset), injection of tumor cells directly into the bloodstream of the animal (to recap distant
seeding), genetically engineered mouse and mice xenografted with patient-derived tumors [64-69] (to mimic
“true” tumors). As to the analysis of circulating miRNAs in these experimental settings, several reports are
available [52,54-56,70,71] [Table 2].

Mice xenografted with human tumor cell lines or patient-derived tumor
The pattern of circulating miRNAs has been extensively studied in mice xenografted with tumor cells and
patient-derived tumors. Different independent studies firmly demonstrated that miRNAs released in the
circulation by the tumor xenograft are distinct from the “background” mouse miRNAs pattern. This is a key
point, since pre-existing miRNAs present in the mouse body fluids are identical in sequence to most human
miRNAs and hence they might be a powerful confounding parameter, possibly altering conclusions and
implications of any circulating miRNA signature. In this respect, the use of laboratory mouse strains has
the advantage that its “background” mouse miRNA pattern is stable and easily quantifiable. Mitchell et al.
[52]
demonstrated that several miRNAs originating from xenografted human prostate cancer cells are present
in the mouse circulation (one of the most interesting being miR-141), and are readily measured in plasma,
allowing a clear distinction between tumor xenografted and control tumor-free mice. Waters et al.
[70]
observed a complex miRNAs dysregulation in the circulation of athymic nude mice subcutaneously injected
with MDA-MB-231 cells. Some miRNAs (such as miR-10b) were undetectable in the circulation, others (miR-
195 and miR-497) were significantly decreased, miR-221 content did not change, and a positive correlation
was observed between miR-497 and miR-195. This study highlighted distinct roles of miRNA subsets in
the circulation and in disease dissemination and progression, all of which may be candidates as molecular
targets for diagnosis as well as design of systemic therapy. Gasparello et al. studied liquid biopsy in mice
[55]
bearing CRC xenografts, demonstrating gateways regulating the levels of circulating tumor-derived miRNAs
(ctmiRNAs), e.g., cell-specific roadblocks that determine whether a given cell xenotransplants releases or
retains a specific miRNA. These roadblocks are often not present in cultured cells, and build “barriers” to
detection in a liquid biopsy format.

Genetically engineered mouse model systems
Genetically engineered mouse models (GEMMs) manipulate target oncogene or tumor suppressor expression
in mice in order to promote tumor development. Transgenic and knockout GEMMs have provided important
models for identifying tumor-associated and metastasis-associated genes that can lead to tumor formation
and disease progression. In addition, GEMMs have been applied to the development of liquid biopsy methods
based on the analysis of circulating microRNAs. Selth et al. performed a global miRNA profiling and
[54]
identified a set of miRNAs exhibiting significantly altered serum levels in transgenic mice models of prostate
cancer (i.e., Transgenic Adenocarcinoma of Mouse Prostate mice). Global miRNA profiling identified 46