Background
Prostate cancer (PCa) is one of the most common malignancies in men [
1]. Despite controversies in the use of prostate specific antigen (PSA) for PCa screening, it is one of the most widely utilized biomarkers, and its clinical use has an undeniable clinical importance [
2]. Nonetheless, PSA alone has not provided accurate diagnostic and prognostic information. Recently, “liquid biopsies” such as circulating exosomes have gained increasing importance [
3]. These vesicles not only function in removing cellular artefacts, but also play an important role in cell-to-cell communications which is due to nucleic acid, protein cargo that is deemed to reflect cell biology of originating tumor cells [
4,
5]. Thus, the spectrum of exosome research is split into isolation from biofluids, functional analysis, and its potential use in clinical assays. Studies have shown that extracellular vesicles including exosomes are a better source of selective miRNAs than the whole blood [
6]. The growing interest in miRNAs, a cargo component of exosomes, is due to its stability as they are either bound to specific proteins e.g. Argonaute2 protein complex (Ago2) or are contained in exosomes protecting them from lysis by RNase in blood. miRNAs (circulating and bound) have been widely investigated in PCa and have promising applicability as prognostic and/or predictive markers [
7]. Cellular stress conditions with microenvironment adaptations involve the release of miRNAs, miRNAs processing as well as changes in miRNAs function [
8]. In cancer, miRNAs act both as oncomirs and tumor suppressors which may equally play a role in treatment response to different stressors including radiation treatment [
9,
10]. Exosomal RNAs mediate genomic instability of recipient cells indicating a stress-induced RNA cargo released due to radiation [
11]. Certain miRNAs (e.g. miR-145) have been found to promote cancer proliferation and radioresistance [
12,
13] which underlines the clinical potential of miRNAs in radiation oncology [
14].
The technical challenges in implementing a robust method for detection of site-specific exosomes in functional studies are well-known [
15]. Many techniques for exosome isolation and recovery of exosomal miRNAs have been published showing that the functional outcomes of exosomes are technique- and sample-dependent (e.g. biofluids, cell culture samples) [
16]. In clinical settings, limited volume of blood may also restrict exosome concentration and miRNA profiling. Moreover, differences in sample storage, processing, RNA extraction and amplification (e.g. qRT-PCR, deep sequencing) have substantial impact on data generation and clinical applicability. miRNAs associated with PCa and radiotherapy response are summarized in our recent paper, although literature is very limited on the topic [
5]. Exosomes therefore represent a promising biological marker for potential optimization of PCa radiotherapy.
Discussion
Recent studies reported the variability in exosome subtypes and cargo composition emphasizing the importance of isolation and characterization methods. In clinical settings, the success of an optimal liquid biopsy workflow is dependent of small sample volumes that allow proper characterization of the exosomal cargo. A better characterization of exosomes origin through molecular and genetic analysis and changes induced by treatment could provide valuable knowledge for treatment personalization. Radiotherapy is a primary curative modality in PCa [
27]. Although previous studies focused on isolation, and characterization of exosomes, variation in paired patient samples before and after radiation has never been investigated. To our knowledge, this is the first study to assess an exosome isolation workflow for PCa radiotherapy. We demonstrated that despite minimal volumes and extraction of exosomal miRNAs, qRT-PCR-based expression analysis is technically feasible.
Although a minimal volume threshold of serum required for optimum yield of exosomes is an important limiting factor, we have isolated exosomes from a volume of 1 ml. A titration study found that the volume of input serum positively correlates to the exosome yield [
28]. In this study, median follow-up sampling time interval was 93 days (min 34; max 148) after radiation which is an important factor to be considered. From biological perspective, quantitative and qualitative variation of exosomes and its RNA molecules would be meaningful in defining their predictive markers for specific therapies, establishing a basis for detection of a temporal relationship [
29,
30]. In general, higher concentration of exosomes in patient samples could indicate radiation specific induction; however, more specific molecules such as DNA damage markers deserve further investigation [
14]. Moreover, exosome vesiculation is proposed to be influenced by multiple factors which may interfere with exosome release and its content [
16]. The method used to isolate exosomes influences the quality and quantity of exosomes [
28]. The PEG has long been used for precipitation of small particles such as viruses that may precipitate exosomes present in the samples (e.g. blood, cell culture supernatant) [
31]. Despite low purity, enrichment of vesicles can be achieved by filtration and certain optimization methods that enriches vesicles significant in quality and quantity for biomarker research [
18]. The input of less serum volumes may cause breaking of vesicles in other methods such as ultracentrifugation causing low particle recovery and biased downstream analysis [
32]. The PEG reagent forms polymer for better precipitation of exosomes yet preserves their biological activities to be used in basic and clinical research [
33]. In particular, PEG-based exosome isolation provides consistent measurements which are in general consensus to most extravesicular research findings [
34].
The Nanosight measurements showed consistent data for exosomes that has been corroborated in other studies with conventional ultracentrifugation methods. This suggests that PEG method is a suitable tool in vesicles recovery from serum [
35]. The Nanosight technology relies on laser light scattering microscopy on Brownian motion of the particles providing size-based particle count as well as respective concentration. This might have limitations in precisely capturing exosomes alone excluding noise created by lipoproteins, protein aggregates, and other biological vesicles from serum [
19]. Another criticism of using NTA technology is the evidence of operator handling bias that may influence the accuracy and reproducibility of the measurements [
36]. As the isolation and enrichment of exosomes from biofluids is an elaborate task, technical variations may impact interpretation of results, especially in comparing individual cases. Thus, sample grouping would arguably make more sense in the interpretation of radiation-induced changes. We have stratified our samples before and after irradiation. Patient variability may show endogenous variability in terms of size and distribution of exosomes, an ideal protocol in NTA that is applicable for each and every sample would be difficult to validate, despite certain possibilities to reduce those variation by optimizing NTA software settings [
19,
37].
Our FACS experimental results are limited in outlining one possible approach in exosome characterization workflow, though implication of exosomal surface markers is briefly discussed. The FACS result may lead to a hypothesis that CD9 surface marker is less expressed compared to CD63 in serum exosomes from PCa patients. This may also indicate the exosomal sub-population theory regarding their concentration, heterogeneous surface markers, and contents are influenced by multiple factors (e.g. clinical phenotypes) [
38]. In contrast, studies have also found exosomes representing higher amount of CD9 surface marker in advanced and chemo-resistant PCa compared to others [
39]. Such variation showing selective enrichment of exosomes can also be due to methodological variation used for their isolation and processing [
15,
40]. While NTA technologies are limited to size based discrimination, use of beads (4.5 µm diameter) labelled with surface markers for exosomes make it possible to characterize them by FACS [
41]. In our pilot study, we used Dynabeads
® coated with primary monoclonal antibody specific for human exosomes surface antigen CD63 without knowing the concentration of optimal antibodies required for samples. A standard titration curve using standard exosome concentration will be essential to ensure most correct number of exosomes captured by beads. Heterogeneous exosome populations with varying levels of surface antigen (e.g. CD63, CD9) or their relative expression is important to consider which may allow to correctly interpret biological causes of (sub) population and concentration of exosomes in biofluids [
42,
43]. It is important to note that multiple numbers of exosomes may bind on a bead which may potentially bias the exact number of exosomes in the given volume of specimen. The detection limit of flow cytometers is 200 nm which is the size threshold used to differentiate exosomes from other microvesicles [
44]. Despite certain limitations by FACS, use of magnetic beads solve one important aspect of identifying exosome in biofluids [
43]. Moreover, recent technical upgrades in FACS targeting multiple surface markers may provide desired level of standardization methods that can be widely used for routine exosomes characterization workflow in future [
44,
45].
In subsequent RNA extraction step, other RNA species such as full length 18S and 28S rRNA peaks were not observed in electropherograms which is in congruence to other similar study [
28]. The choice of exosome isolation method was found to selectively enrich miRNA expression during qRT-PCR e.g. miR-16, let-7a [
46]. Due to low sample input volume, the yield of RNA was less and was difficult to measure by Qubit, Nanodrop [
47]. The possible explanations could be due to multiple washing steps in protocol of Total Exosome RNA and Protein Isolation kit that washed away certain amounts of small RNA. The rate of miRNA synthesis at site of cancer and its half-life in biofluids determine their expression level. The turnover of miRNA in circulating exosome is not yet clear although the median half-life of other mRNA molecule is thought to be around 2 min [
48]. Due to multiple reasons, it is challenging to confirm if the initial RNA volumes were comparable between samples, which would affect the downstream process till expression analysis [
49]. A recent publication using mass spectrometry analysis showed small RNAs as major content of serum exosomes derived from colon cancer patients [
50]. Novel approaches for scaling up exosome concentration may further enhance the possibility of recovering higher miRNA concentration in the serum.
In qRT-PCR analysis, there is no consensus in the choice of reference gene derived from exosomal RNA cargo for data normalization [
51‐
53]. We tested RNU48 that has been used in other studies to normalize miRNAs in qRT-PCR analysis but gave “undetermined” result [
54,
55]. RNU48 has been found to be dysregulated in certain cancer types such as breast and head and neck cancers that could introduce bias in the miRNA expression analysis [
56]. Conclusively, the RNU48 was not present in detectable amount by qRT-PCR so we propose it should not be used as a reliable control for exosome study. We resorted in calculating average expression data for each set of samples for each miRNA target; a common approach used to normalization by taking mean Ct values from a set of miRNAs as similar to global mean normalization [
57,
58].
The expression of hsa-let-7a-5p increased significantly post radiation in HR_F (p = 0.037). The hsa-miR-21-5p expression remained the same in the IR group while differed significantly in the HR_F (p = 0.036). The distinct expression of miRNAs in IR versus HR groups may have clinical ramifications (Fig.
6a). Four miRNAs (miRNA-21, miRNA-34a, miRNA-125, and miRNA-126) observed in comparison of PCa to benign prostatic hyperplasia demonstrate the heterogenity in miRNA expression [
59]. With regard to the heterogeneity of expression data, another study found different sets of miRNAs (let-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-18b-5p and miR-25-3p) that discriminated benign prostatic hyperplasia patients from PCa [
60]. While miR-21-5p better distinguished PCa from benign prostatic hyperplasia, exosomal let-7a-5p was found to be differentially expressed in patients with Gleason score ≥ 8 versus ≤ 6 [
6]. The expression of hsa-let-7a family miRNAs is regulated in prostate cancer [
61], and are altered by ionizing radiation [
62]. Exosomal biogenesis still requires important elucidation, therefore its reliability as a disease biomarker depends on the study design, and also on the specific clinical question [
6,
63,
64]. Unless there is a stringent method to define association of exosome yield and its content with specific clinical endpoints, the cross-referencing from previous studies will still raise questions on its biomarker application [
65]. With pilot samples, we demonstrated the feasibility to detect radiation-associated miRNAs in serum exosomes. Given limited sample size and important clinical heterogeneity, further studies in larger datasets are warranted.
Authors’ contributions
BM, DMA, ADP conceived and designed the study. BM performed the experiments, statistical analysis, and was a major contributor in writing the manuscript. DMA, ADP interpreted the patient data and were contributors in writing and reviewing manuscript. All authors read and approved the final manuscript.