1. Introduction
Over the past years we have witnessed a substantial increase in the number of publications focusing on liquid biopsies. These are particularly useful in the context of cancer, as non-invasive means of diagnosis and follow-up [
1]. MicroRNAs are among the various liquid biopsy-based molecular biomarkers showing promise in this field. They are involved in the post-transcriptional regulation of the functionality of genes, and are crucial modulators of several biological processes, including embryonic and germ cell development [
2]. One of the advantages of microRNAs as liquid biopsy-based biomarkers relates to their relative stability in body fluids. Moreover, they can be easily detected and quantified in a cost-beneficial manner, with high sensitivity and specificity [
3].
Germ cell tumors (GCTs) are very diverse, comprising various histological subtypes (the most common being seminomas [SEs] and the several non-seminoma [NS] subtypes), anatomical distributions (both gonadal—testicular and ovarian—and extragonadal tumors, along the midline of the body) and afflicting a wide range of age groups (pediatric, young-adults and even old-adults) [
4]. Their most fascinating characteristic is that they are developmental cancers: each tumor entity resembles a phase of embryonic and germ cell development and recapitulates the epigenetic pattern of the respective originating cell [
5,
6]. The main variant based on epidemiological characteristics are the so-called Type II testicular germ cell tumors (TGCTs), also known as germ cell neoplasia in situ (GCNIS)-related GCTs of the testis. They are the most common neoplasms among young-adult men in Western civilization, but are also amongst the most curable solid cancers, which could make one assume there is not much more to improve in this field [
7]. However, precisely because of this, both patients and clinicians face novel unexpected challenges, including risk of overtreatment, exposing patients to unnecessary long-term side effects of chemo- and radiotherapy; and also insecurity about stratification of patients to different follow-up and treatment protocols [
8,
9,
10,
11]. The existing biomarkers used nowadays in the clinic (alpha fetoprotein [AFP], human chorionic gonadotropin subunit β [β-HCG] and lactate dehydrogenase [LDH]) are informative, but show limited utility in daily practice to respond to all these issues; therefore, better biomarkers for the disease are needed [
12,
13,
14].
From the biological perspective over these neoplasms, microRNAs emerge as promising biomarkers [
15]. In fact, a set of (embryonic) microRNAs (including the miR-371/373 cluster and the miR-367) have been pinpointed by miR-array to be actively involved in (T)GCT biology [
16,
17,
18], and have proved their value in the past years as biomarkers of (T)GCTs in a multitude of studies with various designs, focusing mainly on type II TGCTs, but also extending to type I pediatric and extragonadal tumors [
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30]. Initial studies consisted mainly of reports of patients where microRNA determination was pursued [
30] and proof-of-principle works with limited number of subjects included [
31,
32]. Given the promising results, larger studies were conducted, retrospective and more recently also prospective, and aimed at solving relevant clinical questions in the field [
22,
33]. In these works, miR-371a-3p was demonstrated to be the most remarkable biomarker [
34,
35,
36], outperforming classical serum markers in their ability to diagnose, follow-up and predict residual disease after chemotherapy in these patients, with sensitivity and specificity over 85–90% [
27,
37,
38]. This microRNA is related to all different histological elements of (T)GCTs, except mature teratoma, for which a proven informative biomarker is lacking so far. This specific microRNA profile in (T)GCTs might also be exploited for treatment purposes, targeting overexpressed oncogenic microRNAs and/or replenishing underexpressed tumor suppressor microRNAs [
39], or even to be used for suicide gene activation [
40].
For any biomarker to be introduced in the clinic, appropriate technical issues should be considered. Novel methodologies may be of use in the future, such as digital droplet PCR and next generation sequencing, which could overcome eventual unspecificity of the assays used, however, the RT-qPCR pipeline validated thus far is attractive for implementation in the clinic, since it represents a relatively low-cost and fast method for testing several patient samples in time, providing clinicians with valuable information. A recent health economic analysis estimated that miR-371a-3p could reduce the costs with germ cell tumor patients follow-up strategy by as much as 44%, especially at the expense of reducing the amount of necessary imaging in microRNA-negative cases [
41]. The value of this standardized pipeline relies on appropriate quality control and normalization [
23,
29]. However, some important issues remain, such as hemolysis content in blood samples, as it can interfere with the detection levels of certain microRNAs [
42,
43], as well as the choice of analysis of serum or plasma. However, the real impact of these matters on specific assays and the best way to approach them is still debatable.
MicroRNAs can be secreted from tumor cells in various ways [
44]. In spite of the data on the putative impact in the clinical setting, microRNA synthesis and secretion dynamics in (T)GCTs are still largely unknown, and a proper characterization of these processes in (T)GCTs (i.e., possible selectivity—
Supplementary Figure S1—and involvement of vesicles) has not yet been tackled. Since these representative models reflect to some extent the biology of these tumors, complementing such in vitro data with further data derived from in vivo pre-clinical models could be extremely valuable to identify the most informative microRNAs for clinical application.
In a recent integrated analysis, Shen et al. [
45] suggested that miR-375 is overexpressed in tissue samples from teratoma and yolk sac tumor (and mixed tumors containing these subtypes); this finding could be extremely useful, particularly in the context of primary diagnosis in cases of pediatric GCT, as well as in the metastatic context after chemotherapy in both pediatric and adult patients. The reason is that detection of (residual) mature teratoma is clinically important and challenging, being the only subtype remaining undetected by the promising miR-371a-3p. However, this data lacks validation in liquid biopsy samples so far.
The aim of this work is to investigate in detail the dynamics of microRNA synthesis and secretion in (T)GCT cell lines, correlating with patterns observed in mouse models, achieving a reliable combined in vitro and in vivo model for identifying the most promising candidate microRNAs. In addition, the impact of pre-analytical variables (hemolysis, choosing serum vs. plasma) on microRNA quantification is investigated. Moreover, the potential role of miR-375 in a liquid biopsy setting, confirming or disproving preliminary data reported on tumor tissues, is performed. This setup will be informative for other disease processes as well.
4. Discussion
Liquid biopsies are progressively conquering their way into the clinical setting. The quest for finding the optimal biomarkers for detection in liquid biopsy setting is on, but many challenges need to be overcome [
55]. In fact, the amount of studies reporting promising biomarkers in limited tissue-based cohorts substantially exceeds the number of liquid biopsy-based validation works. Appropriate control samples, sufficiently large cohorts, pre-analytical variables and different pipelines for quantification are among the reasons for this, sometimes resulting in studies showing controversial and non-reproducible results. Therefore, there is a need for a biological model of predicting candidate biomarkers specifically to be pursued in liquid biopsy setting.
In our work, the miR-371/373 cluster and miR-367 were indeed amongst the most represented in all cell lines and respective media, underscoring that our in vitro model is able to identify the relevant microRNAs that already proved their value as biomarkers of the disease (
Figure 1A,B). Our data also put in evidence that not all microRNAs are secreted with the same efficiency and that selectivity for retaining inside the cells (possibly fulfilling a biological role) does occur (
Supplementary Figure S1). Indeed, when we look at the whole microRNA profiling of cell lines and respective conditioned media, we notice a significant number of non-secreted microRNAs. This is even more remarkable after excluding the numerous microRNAs already present in the fetal calf serum used for cell culturing (
Figure 1C). This evidences the need for considering appropriate controls for experiments [
56], which is not always done and will result in false positive findings.
The remarkable fast level of presence, in spite of low number of cells, and stability of miR-371a-3p levels in conditioned media suggests that a regulatory mechanism for this microRNA secretion exists, that renders it independent of external conditions (
Figure 2A,B). We hypothesize that a protecting packaging mechanism might contribute to these findings. Indeed, our observations (
Figure 2C) suggest that miR-371a-3p is present in exosomes (like demonstrated for other microRNAs [
57]), given its consistent presence at similar levels in the whole and exosomal microRNA fractions; this finding should be confirmed in further studies and for other relevant targets and cell lines. Also, the amount of EVs was found to associate better with miR-371a-3p levels than the simple number of cells in culture, further strengthening this idea. Levels of miR-371a-3p were lower in exosome fractions isolated via CD63+ immunoprecipitation, in line with previous findings [
58] showing that CD63 is not equally present in all exosome fractions. All in all, our data shows that the protocol will not increase in value by including an exosome purification step. This finding expands our knowledge on the mechanisms of secretion of these relevant microRNAs specifically in GCTs.
Hemolysis is a factor known to influence microRNA detection in serum/plasma samples [
59]. Several methodologies for measuring hemolysis burden in serum samples have been described, with the so-called “miR-23a/451a ratio” being reported as the most accurate [
42]. However, the work of Shah and collaborators differs from ours in various ways, including cohort size, microRNA extraction and quantification methods. Hence, it is fair to assume that the same cutoffs determined by the authors are not necessarily applicable in our workflow. Indeed, hemolysis did not show significant direct impact on the microRNA isolation procedure (denoted by ath-miR-159a) nor on the specific target assay miR-372a-3p, which contrasts with the findings of Myklebust and coworkers [
43]. An effect on the normalizer miR-30b-5p levels was depicted; however, such effect was minor and seen mainly at the expense of cases with severe hemolysis, with visual scores of 4–5 (
Figure 8), which were absent in our study cohorts. When applying the new miR-23a/451a cutoff determined by us in a second validation cohort, we found no significant differences in distribution of Ct values of spike-in, normalizer nor the specific targets miR-371a-3p/miR-375. Importantly, we believe the major reason for the little impact of hemolysis on specific targets in our study is the distinct microRNA isolation procedure: we have performed a magnetic bead capture of microRNAs, which are isolated and purified from the serum contents, eliminating the potentially harmful effects of molecules such as hemoglobin or heparin, which are known to inhibit the PCR reaction [
60,
61,
62,
63]. Indeed, we have witnessed this effect in RNA isolated from kidney preservation fluids (described in [
54]) containing heparin contamination, a commonly used anticoagulant. Our new results again demonstrate that in the bead-based capture no inhibitory effect of heparin was observed and heparinase 1 digestion did not reduce the Ct values of both endogenous and spiked-in microRNAs (
Supplementary Figure S12). This strengthens the advantages of this established pipeline when aiming to quantify microRNAs in liquid biopsy setting.
Evaluation of matched serum/plasma samples from the same patients depicted significant differences in the amount of the commonly used endogenous reference microRNA, miR-30b-5p, with plasma samples showing significantly lower Ct values. Significant differences in the amount of target microRNAs were also depicted (
Figure 9). This knowledge is of particular importance as it may be a potential problem in mixed cohorts comprising both serum and plasma samples, again supporting the idea that mixed cohorts of both body fluids are not advisable. We hypothesize this could be due to different compositions of both fluids, both a combination of the relative proportion of fluid volume and the amount of clotting factors present in plasma samples, which might result in microRNAs sticking to them, hence escaping quantification [
64]. The higher total volume of serum could explain the higher Ct values (i.e., lower levels) of miR-30b-5p when compared to plasma. The reason for observing higher Ct values for miR-371a-3p in plasma is not known, although we stress that it follows the same tendency observed for ath-miR-159a (although the latter was not statistically significant) and hence might reflect slight differences in the microRNA purification step. Overall, these results suggest that both plasma and serum can be reliably used for microRNA analyses, as previously demonstrated, but should not be compared to one another in mixed cohorts.
MicroRNA profiling of CSF samples deemed to be negative for neoplastic disease and inflammatory disease have not been reported thus far. Murray and collaborators [
29] described a pipeline for quantifying microRNAs in CSF samples from pediatric GCT patients, including in their cohort four CSF samples. As controls of the experiments, five sera samples from pediatric individuals (three females and two males) were included. Our data further extends knowledge on this matter; when comparing the microRNA profiling of both normal CSF and sera samples (control subjects) we observed that a much higher proportion of tested microRNAs were absent in CSF samples when compared to sera. In CSF, 80% of microRNAs were absent in all four samples, compared to 34% in sera. Our data indicates significant differences between microRNAs present in the normal bloodstream compared to CSF, possibly due to their difference in origin [
65,
66]. All in all, our data stress the specificity of finding elevated levels of clinically relevant microRNAs in CSF samples for diagnostic purposes, including the use of normalization for which miR-30b-5p is not appropriate, and an alternative must be determined.
Moreover, one must take into account the potential for microRNA decreasing (and its timing) after surgery. Our data on microRNA measurements after orchiectomy suggests that, indeed, the most reliable microRNA among the tested ones is the miR-371a-3p, which shows a steady decrease after orchiectomy and exhibits a very short half-life (<4h in our study), showing a superior profile compared to other targets (
Figure 5), in line with earlier findings [
25]. The fluctuations observed (in one single patient) for miR-372-3p and miR-367-3p are likely due to unspecificity of the assays used, as demonstrated by our experiment when various RT-primers were combined in pools. miR-372-3p is in a microRNA cluster together with miR-371a-3p and miR-373-3p, so cross-reaction is plausible. For miR-375 no data is available to suggest unspecificity; however, this target was found anyways to be non-informative in all the settings tested for germ cell tumors, and fluctuations may simply reflect this issue.
Similar to our in vitro findings, the in vivo mouse model further confirms the ability of identifying relevant biomarkers of the disease. Indeed, the miR-371/373 cluster and miR-367 (among others) were predicted to be most informative in plasma samples for discriminating malignant disease (
Figure 3A). One of the major quests in the field of GCTs is the finding of a biomarker specific of the teratoma histology, namely one that allows the detection of residual mature/benign teratoma, for which treatment approach might differ. When focusing on microRNAs positive in these tumor xenografts, indeed most of them are not amenable to detection in plasma; however, a handful of microRNAs are pointed out by our model, namely the miR-885-5p, which could be of interest in this context (
Figure 3B). Again, the relevance of using appropriate controls is underscored by the number of candidate microRNAs discarded due to their expression in normal mouse tissues/plasma (mouse-specific microRNAs instead of tumor-specific microRNAs). Based on this we pursued targeted analysis attempting to validate these markers in clinical samples. On the post-chemotherapy RPLND patient cohort, all three microRNAs were in the detection range, with no significant differences in relative levels among cases of viable tumor, teratoma or solely fibrosis/necrosis, which limits the value of these markers in this specific clinical scenario. Detection of these markers at similar levels in both teratoma and fibrosis/necrosis cases is intriguing and deserves further investigation in future studies. miR-885-5p has been shown to be a strong activator of the p53 pathway, inducing apoptosis, senescence and cell cycle arrest [
67]. This makes this target very appealing from a biological point of view, since p53 pathway is activated specifically in the (miR-371a-3p-negative) teratoma [
68], as opposed to the other (malignant) GCT components, which show clear upregulation of miR-371a-3p, leading to inactivation of the p53 pathway [
69]. This shift in microRNA expression and impact on p53 pathway might shed light into the distinct biology and clinical behavior of these tumors. This is also in line with our findings of absence of miR-885-5p in NCCIT cells, which show absence of functional p53, while it is present in NT2 cells, where p53 is active. Also, tumor cells in necrosis (and apoptosis) after chemotherapy might be a source of miR-885-5p, explaining its expression in all RPLND samples, as some degree of necrosis/apoptosis is always present in every post-chemotherapy metastatic mass. The finding of miR-885-5p to associate with fetal growth and sperm count also seems to demonstrate an association with development, which fits the developmental model for GCTs [
70,
71]. Meanwhile, miR-448 has been described as inhibiting cell proliferation and invasion in several tumor models [
72,
73,
74], but its role in this specific context is still unclear. However, despite being of limited use in this specific discrimination, these microRNAs were found to be significantly upregulated in serum samples of patients with teratoma histology when compared to normal males, allowing for a good discrimination among the two groups, which fully confirms the prediction of the in vivo model. Nevertheless, our attempt to validate these biomarkers was pursued in the specific context of post-chemotherapy RPLND masses and in a limited number of subjects, deserving for sure further validation in larger studies, to accurately assess the discrimination performance of such microRNAs, including the ability to follow-up patients with primary teratoma of the testis, both adult and pediatric. Inability to detect the teratoma histological subtype has been one of the few critics appointed to miR-371a-3p as a disease biomarker. Specifically, miR-375 was suggested to solve this gap, however shown in this study not to fulfill the necessary requirements for this purpose (which is in line with recently published data [
75]), although miR-885-5p and miR-448 might be informative. In addition, this is of relevance as well in the context of regenerative medicine, in which possible formation of (benign and malignant) tumors is a significant limitation in clinical application [
50].