Non-Exosomal and Exosome-Derived miRNAs as Promising Biomarkers in Canine Mammary Cancer

Canine mammary cancer (CMC), similar to human breast cancer (HBC) in many aspects, is the most common neoplasm associated with significant mortality in female dogs. Due to the limited therapy options, biomarkers are highly desirable for early clinical diagnosis or cancer progression monitoring. Since the discovery of microRNAs (miRNAs or miRs) as post-transcriptional gene regulators, they have become attractive biomarkers in oncological research. Except for intracellular miRNAs and cell-free miRNAs, exosome-derived miRNAs (exomiRs) have drawn much attention in recent years as biomarkers for cancer detection. Analysis of exosomes represents a non-invasive, pain-free, time- and money-saving alternative to conventional tissue biopsy. The purpose of this review is to provide a summary of miRNAs that come from non-exosomal sources (canine mammary tumor, mammary tumor cell lines or canine blood serum) and from exosomes as promising biomarkers of CMC based on the current literature. As is discussed, some of the miRNAs postulated as diagnostic or prognostic biomarkers in CMC were also altered in HBC (such as miR-21, miR-29b, miR-141, miR-429, miR-200c, miR-497, miR-210, miR-96, miR-18a, miR19b, miR-20b, miR-93, miR-101, miR-105a, miR-130a, miR-200c, miR-340, miR-486), which may be considered as potential disease-specific biomarkers in both CMC and HBC.

Life 2022, 12, 524 2 of 34 from 1% to 6% and 13%, respectively [8]. Ovarian hormones (estrogen, progesterone) may lead to carcinogenesis and mammary hyperplasia. Thus, ovariohysterectomy is precautionary for tumor development, and its timing seems to be crucial [10,11]. A female dog of any breed castrated before the first ovarian cycle has a 0.5% chance of developing a tumor. If the bitch is spayed just after or at any subsequent ovarian cycle, the risk of mammary gland cancer increases from 8% to 26% [9,10]. Additionally, the study of Schneider et al. demonstrated that ovariohysterectomy of bitches after their second estrum had no preventive impact against the development of malignant tumors [10].
Canine studies also indicated that obesity is another major risk factor for mammary tumor development, especially if present early in a dog's life [12,13]. The study of Sonnenschein et al. demonstrated that a thin physique reduced the risk of mammary cancer among spayed dogs by 99%, and non-spayed dogs by 40% [12]. The influence of the diet was also studied. Dogs on a homemade diet with high-red meat portions were at a higher risk of developing mammary dysplasia and tumors compared to a commercial diet [13]. Therefore, nutritional factors, operating especially early in life, also have etiological importance to canine mammary cancer (CMC) development.

Classification System
Mammary glands are a frequent location for tumor development and, as in other types of cancer, canine mammary tumors may be benign or malignant [14]. Since approximately half of mammary tumors in dogs are malignant with a high percentage of mortality if not treated in time, there is no doubt that canine mammary neoplasia represents a serious clinical issue [4,15,16]. Histopathology and biopsy remain the cornerstone and the gold standards for the diagnosis and classification of canine mammary tumors [17]. However, the morphological heterogeneity of these tumors, with frequent presence of various cell populations, is challenging when providing an appropriate classification [17]. Nowadays, veterinary pathologists have available two systems of a histological classification scheme for canine mammary tumors: the official histological classification approved by the World Health Organization (WHO) and the Armed Forces Institute of Pathology from 1999 [18] and the international consensus histological classification scheme based on 2011 updates to the WHO HBC parameters proposed by Goldschmidt et al. from 2011 [19]. The latest 2011 system combines various criteria for subtyping mammary tumors by separating benign forms from malignant lesions and determining the tissue of origin (epithelial, myoepithelial, mesenchymal). A comparison of the two classification systems by Canadas et al. demonstrated that the WHO and 2011 classification systems were very similar in terms of the categorization of benign tumors, and both were prognostically relevant by identifying malignant tumors [17]. Therefore, veterinary pathologists should include both classification systems in the diagnosis and classification of canine mammary tumors.
Based on the tissue of origin, mammary gland tumors of purely epithelial origin are malignant carcinomas, such as carcinoma in situ, simple carcinoma (tubular, tubulopapillary, cystic-papillary, cybriform), solid carcinoma, anaplastic carcinoma, ductal carcinoma, complex and mixed type carcinoma [17,18]. However, there are also other special types of malignant epithelial neoplasms (squamous cell carcinoma, adenosquamous carcinoma, mucinous carcinoma, lipid-rich carcinoma, spindle cell carcinoma, and inflammatory carcinoma) [18,19]. Tubular carcinoma (adenocarcinoma) is the most common type of mammary gland tumor in dogs [19,20]. Mesenchymal neoplasms are sarcomas (osteosarcoma, fibrosarcoma, chondrosarcoma, liposarcoma, hemangiosarcoma, and others), with osteosarcoma as the most frequent mesenchymal neoplasm of the canine mammary glands [19]. However, some of them have mixed histology consisting of a combination of epithelial and myoepithelial or mesenchymal tissue (complex carcinoma, carcinosarcoma, and benign mixed tumors) [19]. Benign mammary tumors are mostly simple and complex adenomas, fibroadenomas, myoepithelioma, ductal adenoma and ductal papilloma [18,19].
The cytology of canine mammary tumors can be another approach in diagnostics, but it should be taken into account carefully, because it offers many false results due to lesions

Treatment
Even though the improved health of dogs, as a result of better quality and more easily reachable veterinary care, treatment, and better nutrition, allows dogs to live longer, the incidence of cancer in dogs is constantly increasing [20]. Surgery consisting of the removal of the affected (cancerous) glands and local lymph nodes is currently the only efficient treatment and can be curative in many dogs [40,41]. In malignant cases, chemotherapy and radiotherapy are applied [33,42]. However, this aggressive approach is expensive and limited, with no definitive data [33,42]. No protocol for chemotherapy in female dogs affected by mammary gland tumors has thus far been standardized [41]. In addition, chemotherapy in dogs has not been proven to be as effective in the treatment of mammary gland cancers as it is in women [42,43]. Standard chemotherapeutics, such as docetaxel and doxorubicin, have not demonstrated dramatically improved overall survival times [44]. Thus, at this time, no effective systemic treatment options for dogs with mammary tumors are available. Because of the limited treatment possibilities, early diagnosis of cancer, Life 2022, 12, 524 5 of 34 evaluation of the cancer progression, and tumor response to chemotherapy can increase the survival of dog patients. Biomarkers represent a valuable tool in cancer research since they offer many applications, such as screening, differential diagnosis, prognosis determination, prediction to treatment, and disease progression monitoring [45,46].

MicroRNAs as Potent Biomarkers
A biomarker is generally defined as a quantifiable measure of a normal biological process or pathological process or as a response to a therapeutic administration [47]. In other words, a biomarker offers information about the actual condition of a living organism. Changes in biomarkers expression levels, concentrations or structure may indicate the onset, progression or regression of some disorder in the body [48]. Biomarkers can be represented by nucleic acids (DNA or RNA) [49], peptides [50], proteins [51], lipids [52] or metabolites [53].
MicroRNAs (miRNAs or miRs) are becoming potential non-invasive cellular and molecular biomarkers for the prediction, diagnosis, prognosis, and therapeutic targets for various types of cancers. Several studies have thus far confirmed the relevance of miRNAs in cancer-associated processes, including proliferation, differentiation, invasion, angiogenesis, metastasis, apoptosis, and drug resistance (reviewed in [54][55][56][57]).

Biogenesis and Function
miRNAs are short (18-22 nucleotides), highly evolutionary conserved members of small non-coding RNAs discovered in 1993 in a model organism Caenorhabditis elegans [58,59]. The miRNA arises as a transcription product of non-coding regions or introns by RNA polymerase II [60]. Still in the nucleus, the resulting hundreds of nucleotides long primary miRNA (pri-miRNA) is subsequently cleaved by the endonuclease enzyme Drosha (RNAse III) and its cofactor microprocessor complex subunit DGCR8 (DiGeorge syndrome critical region gene 8, also known as Pasha), giving the precursor miRNA (pre-miRNA) [61]. Pre-miRNA has a hairpin and loop-shaped secondary structure with 80-100 nucleotides [62,63]. This pre-miRNA is transported from the nucleus into the cytoplasm by the exportin-5 protein and the Ran-GTP complex [64]. Here, the hairpin region of the pre-miRNA is processed by cytoplasmic ribonuclease Dicer into an 18 to 22 nucleotide long double-stranded miRNA duplex which contains two 5' phosphorylated sequence strands with 3 overhangs, named the mature miRNA guide strand and complementary passenger strand [65,66]. The miRNA duplex is unwinded into a single-stranded mature miRNA guide strand (depicted with black color in Figure 1), while the second passenger strand is degraded (depicted with red color in Figure 1) [67,68]. The mature miRNA strand binds to Argonaute 2 (Ago2) protein and other RNA-binding proteins (e.g., protein kinase RNA activator, PACT; trinucleotide repeatcontaining gene 6A protein, TNRC6A; transactivation response RNA-binding protein, TRBP) to form an RNA-induced silencing complex (RISC) that regulates the translation of target messenger RNA (mRNA) [67]. In addition to the transcription repression within the cell, the mature miRNA can be also secreted from the cell as free (circulating) miRNA or intracellularly packed into the extracellular vesicles (EVs), such as exosomes (or small extracellular vesicles, sEVs) or microvesicles (or medium/large extracellular vesicles; m/lEVs) [69,70]. Mature miR-NAs are selectively incorporated into the sEVs (exosomes) or enwrapped with microvesicles during their biogenesis (in process of early endosome inner membrane budding), as explained in Section 3.1, and subsequently, released to the extracellular milieu [70]. Such EV-packed miRNAs are delivered, through the EVs, to other target cells, where the miRNAs regulate their cognate target genes at the transcriptional level [69,[71][72][73][74]. Within the cell, mature miRNAs are associated with RNA-binding proteins, such as Ago2, which protect free miRNAs from degradation by RNases after their release from the cells to the extracellular environment [75]. Free miRNAs are presented in different biofluids (such as blood plasma or serum [76,77], urine [78], breast milk [79], saliva [80], tears [81], or cerebrospinal fluid [82]) [75]. However, the precise mechanism of how free miRNA is released from cells is still not clear [69,70]. The process of miRNA biogenesis is summarized in Figure 1. The miRNAs play a key role as negative post-transcriptional gene regulators in the safeguarding of all biological processes of multicellular organisms, including cell-cycle control, cell proliferation, differentiation, migration, metabolism, and apoptosis [83]. Regulatory action is mediated by the hybridization of miRNA to the 3 -or 5 -untranslated regions (UTRs) [84,85], or the open reading frame (ORF) [86] of the target mRNAs, resulting in the suppression of the expression of the protein-coding genes either by translational repression, mRNA degradation or both [87,88]. More specifically, perfect base complementary leads to mRNA degradation, while non-perfect (partial) base complementarity results in translation impairment [89].
ogenesis (in process of early endosome inner membrane budding), as explained in Section 3.1, and subsequently, released to the extracellular milieu [70]. Such EV-packed miRNAs are delivered, through the EVs, to other target cells, where the miRNAs regulate their cognate target genes at the transcriptional level [69,[71][72][73][74]. Within the cell, mature miR-NAs are associated with RNA-binding proteins, such as Ago2, which protect free miRNAs from degradation by RNases after their release from the cells to the extracellular environment [75]. Free miRNAs are presented in different biofluids (such as blood plasma or serum [76,77], urine [78], breast milk [79], saliva [80], tears [81], or cerebrospinal fluid [82]) [75]. However, the precise mechanism of how free miRNA is released from cells is still not clear [69,70]. The process of miRNA biogenesis is summarized in Figure 1.
The miRNAs play a key role as negative post-transcriptional gene regulators in the safeguarding of all biological processes of multicellular organisms, including cell-cycle control, cell proliferation, differentiation, migration, metabolism, and apoptosis [83]. Regulatory action is mediated by the hybridization of miRNA to the 3′-or 5′-untranslated regions (UTRs) [84,85], or the open reading frame (ORF) [86] of the target mRNAs, resulting in the suppression of the expression of the protein-coding genes either by translational repression, mRNA degradation or both [87,88]. More specifically, perfect base complementary leads to mRNA degradation, while non-perfect (partial) base complementarity results in translation impairment [89]. Biogenesis and release of microRNA (miRNA) and exosomes. The miRNA initially originates as primary miRNA (Pri-miRNA). Pri-miRNA is cleaved into the precursor miRNA (Pre-miRNA) by the Drosha enzyme and its cofactor Pasha [61,62]. Exportin-5 protein and Ran-GTP complex transport the pre-miRNA into the cytoplasm, where it is processed into the double-strand miRNA duplex by the action of a Dicer endonuclease [64][65][66]. One of the strands is degraded (socalled passenger strand; depicted with red color) and the second, mature miRNA strand (also known as guide strand; depicted with black color) is loaded into the RNA-induced silencing complex (RISC) by the binding to RNA-binding proteins (Argonaute 2, Ago2; trinucleotide repeat-containing gene 6A protein, TNRC6A; transactivation response RNA-binding protein, TRBP) [67,68]. The mature miRNA strand is then guided to the target messenger RNA (mRNA) to either degrade Figure 1. Biogenesis and release of microRNA (miRNA) and exosomes. The miRNA initially originates as primary miRNA (Pri-miRNA). Pri-miRNA is cleaved into the precursor miRNA (Pre-miRNA) by the Drosha enzyme and its cofactor Pasha [61,62]. Exportin-5 protein and Ran-GTP complex transport the pre-miRNA into the cytoplasm, where it is processed into the double-strand miRNA duplex by the action of a Dicer endonuclease [64][65][66]. One of the strands is degraded (so-called passenger strand; depicted with red color) and the second, mature miRNA strand (also known as guide strand; depicted with black color) is loaded into the RNA-induced silencing complex (RISC) by the binding to RNA-binding proteins (Argonaute 2, Ago2; trinucleotide repeat-containing gene 6A protein, TNRC6A; transactivation response RNA-binding protein, TRBP) [67,68]. The mature miRNA strand is then guided to the target messenger RNA (mRNA) to either degrade (perfect base complementarity) or inhibit the mRNA translation (partial base complementarity) [89]. The mature miRNA can be also secreted from the cell as free miRNA bound to RNA-binding proteins or incorporated, within the cell, into the extracellular vesicles (EVs), specifically exosomes and microvesicles [69,70]. Exosomes or small extracellular vesicles (sEVs; <200 nm) [90] are produced within the cells starting with the formation of early endosomes by cell membrane invagination [91][92][93]. The inner membrane budding of the early endosome leads to the maturation of the multivesicular bodies (MVBs) [91][92][93]. Some of MVBs are directed to lysosomes for degradation, while others are released to the extracellular space as exosomes after fusion with the plasma membrane [94,95]. Microvesicles or medium/large extracellular vesicles (m/lEVs; >200 nm-1000 nm) [90] are formed in the process of outward plasma membrane budding [96,97]. Apoptotic bodies (>1000 nm), the largest group of EVs, are released from the cells undergoing apoptosis by plasma membrane blebbing [90,98,99]. An original figure was created using Inkscape v1.1.2 software.

The Role of miRNAs in Cancer
Since gene regulation at the transcriptomic level does not require the high complementarity of miRNA with the mRNA sequence, a single miRNA may target several mRNAs, and aberrant miRNA expression has the potential to considerably alter the expression level of several hundred transcripts [100,101]. Dysregulation of miRNAs is particularly prevalent in cancer, where the genetic instability of tumors (such as amplifications, deletions, mutations, epigenetic changes or polymorphisms) leads to altered miRNA expression profiles promoting oncogenesis [102,103]. Downregulated and deleted miR-15a and miR-16-1 in patients with chronic B-cell lymphocytic leukemia were firstly reported as altered miRNAs, leading to the onset, progression, and dissemination of cancer [104]. Subsequently, the interface between overexpression or ablation of miRNA and cancer development was exhibited in mouse models [102,105]. Nowadays, it is known that more than half of miR-NAs are located in cancer-associated genomic regions [106]. Generally, miRNAs involved in cancer are either tumor suppressors or oncogenes, depending on the expression levels [107]. Overexpressed miRNAs, oncogenes, with a crucial role in the initiation and progression of cancer, have been termed oncomiRs [108]. As of February 2022, more than 40,000 free-full peer-reviewed articles dedicated to the investigation of the role of miRNA in cancer by diverse experimental approaches are available in the PubMed depository (https://pubmed.ncbi.nlm.nih.gov/?term=mirna+cancer&filter=simsearch2.ffrft (accessed on 1 February 2022)).

Non-Exosomal miRNA-Based Biomarkers of Canine Mammary Cancer
As of February 2022, 502 precursors and 453 mature miRNAs have been identified in the canine genome (miRBase database; https://www.mirbase.org/summary.shtml? org=cfa (accessed on 1 February 2022)) and most of them have been altered in CMC. As was discussed above, CMC and HBC demonstrate comparable clinical and pathological characteristics. Similarities in the miRNA expression pattern between canine mammary and human breast neoplasia have also been described [109] and several oncomiRs have been found to be highly conserved between dogs and humans [110,111]. These findings are not surprising, since dogs and humans share not only the same environment but also analogous diseases [112]. Moreover, considering the similarities between dogs and humans at the genetic level, miRNAs may target genes conserved between both. Aberrant expression of miRNAs implicated in cancer development, progression or metastasis may serve as a useful biomarker for diagnostic or prognostic purposes and, therefore, represent a target for therapy development [102].
Here, we review the most relevant miRNAs not packed into exosomes (hereinafter called non-exosomal) found in CMC studies in relation to the biomarkers for future clinical applications and compared their incidence in HBC. We found 11 articles related to the topic. According to the analysis carried out for this review, here the term "non-exosomal" is referred to miRNAs which come from sources such as mammary tumors [110,111,[113][114][115], tumor mammary cell lines [116][117][118] or canine blood serum [119][120][121] using commercial kits.

miR-21
It is assumed that overexpression of miR-21 is a hallmark of carcinogenic cells and may serve as a common signal of pathological growth or cell stress [122]. The miR-21 is highly conserved and one of the most abundant miRNAs expressed in multiple mammalian cell types [122,123]. Physiologically, miR-21 regulates processes connected to cell growth, migration, and invasion [124]. In carcinogenesis, miR-21 acts as the oncomiR through the inhibition of tumor cell apoptosis [110,125,126]. Except in the study of Boggs et al. [110], the upregulated expression of miR-21 in canine benign or malignant tumors in comparison to normal glands was observed in several canine mammary studies [113][114][115]119,120]. The elevated expression of miR-21 in female dogs with mammary tumors is in correlation with progressive clinical stage and poor prognosis [119]. Thus, the level of miR-21 expression may be useful for distinguishing between bitches with mammary tumors (benign or malignant) and healthy ones (without mammary tumors) [119]. Moreover, increased expression of miR-21 in metastasis carcinoma (5.05-fold) compared to normal mammary gland makes it a good metastasis biomarker [114]. Regarding HBC, the altered expression of miR-21 was associated with increased cell proliferation, colony formation, migration, invasion, metastasis, angiogenesis, advanced tumor stage, lymph node metastasis, and poor patient survival [127][128][129][130][131][132]. Blocking miR-21 expression inhibits tumor growth and metastasis [133]. As miR-21 is one of the most upregulated miRNAs in HBC, it was postulated that targeting miR-21 by miR-21 inhibitors (anti-miR-21) as post-transcriptional gene silencing agents may have a therapeutic potential [134][135][136]. It follows that miR-21 represents a sensitive non-invasive biomarker for cancer screening, progression, and detection in CMC as well as in HBC.

miR-29b
Another non-invasive biomarker for diagnostic and prognostic purposes for various types of cancer, including mammary cancer, can be miR-29b [137,138]. As a member of the miR-29 family together with miR-29a and miR-29c, miR-29b appears to have a crucial effect on mammary tumors by regulating multiple cancer-related processes essential for tumor development, such as proliferation, apoptosis, metastasis, fibrosis, angiogenesis, proteolysis or collagen remodeling [139]. However, the exact role of miR-29b in cancer remains controversial, as it has been declared as an oncomiR and tumor-suppressor [138][139][140][141][142]. The differential expression of miR-29b has also been noted in CMC. Together with the study of Boggs et al. [110], the upregulated expression of miR-29b was observed in canine SNP cell line (4.0714-fold) [116] or serum samples from canine mammary carcinoma dogs (2.78-fold) [121]. In contrast, a significant downregulation of miR-29b expression in metastasizing and non-metastasizing mammary tumors was observed in the studies of Jain et al. [119], Bulkowska et al. [113], and von Deetzen et al. [114]. Due to the altered expression of miR-29b in a metastatic group in comparison with benign tumors, miR-29b may present another valuable biomarker for metastasis [113]. An inconsistent downregulated [143] or upregulated [144] expression pattern of miR-29b was also observed in HBC, wherein this was connected with proliferation, migration, impaired apoptosis, increased tumor cell migration, and invasion.

miR-141
The very first evidence of comprehensive expression profiles of the 277 investigated miRNAs from the canine genome, which were evaluated using a quantitative polymerase chain reaction strategy in cell lines derived from female dogs of different breeds with spontaneous mammary carcinomas or adenocarcinomas (CMT12, CMT27, and CMT28), revealed miR-141 to be a potent oncomiR [117]. In this study, miRNA-141, a member of the miR-200 family, was experimentally validated to target 3 -UTR of a tumor suppressor INK4 (inhibitor of CDK4), a member of the INK4/CDNK2 family of tumor suppressor genes, through the direct correlation between the overexpression of miR-141 and the target mRNA p16/INK4A in cell lines CMT12 and CMT27 [117]. Significant high expression levels of miR-141 are strongly associated with highly aggressive breast carcinomas (grade III) when compared to grade II breast cancer. ROC curve analysis revealed the diagnostic and prognostic utility of miR-141 in the discrimination of malignant from benign breast tissues (ROC-AUC = 0.97). Moreover, high expression of miR-141 is associated with worse overall survival (OS) in breast cancer patients (HR = 1.43, 95% CI = 1.17-1.74, p = 0.00037; Life 2022, 12, 524 9 of 34 among 1262 patients) [145]. Additionally, upregulation of miR-141 promotes the migratory and invasive abilities of an aggressive triple-negative breast cancer cell line MDA-MB-231 through regulation of the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) signaling pathway by increased secretion of vascular endothelial growth factor A (VEGF-A) and expression of integrin-αV [146]. Together, all these data highlight the role of miR-141 as a valuable biomarker with potential clinical applications in CMC as well as HBC.

miR-429 and miR-200c
The study of Lutful Kabir et al. reported another group of miRNAs to be altered in both canine mammary and human breast tumors [117]. The miR-9, miR-155, miR-200a/b, and miR-429 were overexpressed, whereas miR-1, miR-133a/b/c or miR-214 were found to be downregulated in canine cell lines CMT12, CMT27, and CMT28 [117]. In particular, miR-429 and miR-200c were found to be highly upregulated (>1000 fold and 100-150 fold, respectively) and predicted to target the tumor suppressor ERBB receptor feedback inhibitor 1 (ERRFI1) mRNA [117]. Thus, both miRNAs act as oncomiRs in CMC [117]. Comparable to HBC, miR-429 was also described as an oncomiR that affects the hypoxia-inducible factor 1-alpha (HIF1α) pathway by targeting VHL mRNA [147]. The overexpressed miR-429 in breast cancers with amplified human epidermal growth factor receptor 2 (HER2+) was responsible for the increased proliferation and migration of breast cancer cells, while the silencing of miR-429 had an impact on tumor growth postponement [147]. In contrast, miR-200c was reported as a tumor suppressor in breast cancer tissue and cell lines where suppress the cell proliferation by targeting KRAS mRNA [148], contributes to the paclitaxel resistance by targeting (sex-determining region Y)-box 2 (SOX2) transcriptional factor [149], or inhibits the metastasis of triple-negative breast cancer [150]. Since both miRNAs are involved in the tumorigenesis and progression of a variety of cancers, they may represent potent biomarkers in CMC and HBC.

miR-497
Tumor-suppressor miR-497 family members (miR-497, miR-195, miR-15, and miR-16) were found to be downregulated in canine mammary cell lines [118]. Downregulation of miR-497 was also observed in the CMT1211 and CMT7364 cell lines compared to primary canine mammary gland cells [118]. Transfection of miR-497 mimic and inhibitor into the canine mammary tumor cells showed that overexpression of miR-497 significantly inhibited cell proliferation and migration, and increased the apoptosis in the CMT1211 and CMT7364 cell lines [118]. The observed negative correlation between miR-497 and the expression of interleukin-1 receptor-associated kinase-like 2 (IRAK2) suggested IRAK2 as a functional target gene of miR-497. The suppression of IRAK2 mRNA by the overexpressed miR-497 induced apoptosis by inhibiting the activation of the pro-survival NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway [118]. This study demonstrated that miR-497 inhibits cancer cell growth, with the suggestion of the miR-497/IRAK2/NF-kB axis as a potential mechanism for CMC development [118]. Therefore, miR-497 was suggested as a diagnostic biomarker and therapeutic target in CMC [118]. These findings are consistent with HBC, where miR-497 was among the most prominently downregulated miRNAs [151]. Several studies have demonstrated that overexpression of miR-497 inhibited the proliferation, invasion, metastasis, angiogenesis or cell cycle of cancer cells, and induced apoptosis in HBC by targeting Bcl-2-like protein 2 (Bcl-w) [152], B-cell lymphoma 2 protein (Bcl-2) [153], yes-associated protein 1 (YAP1) [154], HIF-1α [155], or cyclin E1 [156] mRNA.
The expression levels of miR-10b, miR-125b, miR-136, and let-7f in particular gradually decreased from normal mammary tissue, through benign tumors and non-metastatic malignant tumors, to metastatic tumors [113]. These findings are of great predictive importance for the course of a disease and, therefore, altered miRNAs may constitute molecular markers of metastasis.
On the other hand, the expression level of miR-143 in non-metastasizing mammary carcinoma [114] or the canine SNP cell line established by Osaki et al. [116] was higher in comparison to normal mammary gland tissue (2.70-fold and 1547.9-fold, respectively). Likewise, miR-203 expression was downregulated in benign tumors compared to a healthy control group [113]. Such discrepancies in the expression level of one particular miRNA may be a result of changes in gene expression in the tumor, different tumor phenotypes or even different data analyses used to evaluate miRNA expression [113].

miR-210
Some miRNAs are expressed at different stages of malignancy [114]. For example, miR-210 was found to be present in malignancies, such as adenoma, non-metastasizing carcinoma, metastasizing carcinoma, and metastatic tissue with gradually increased expression (7.01-fold, 10.41-fold, 10.72-fold, and 19.63-fold respectively) [114]. As explained by the authors of the study, miR-210 has been termed a hypoxamir due to its upregulation as a result of hypoxia in tissues and it mediates the metabolic adaptation to anaerobic conditions [114,157]. Therefore, rising expression during the progression of malignancy may be a result of increased hypoxia in tumor growth. Since miR-210 is associated with the formation of capillary-like structures [158], the author also hypothesized its role in metastasis by enhanced angiogenesis. This makes miR-210 another potential diagnostic marker in malignancies [114]. Higher expression of miR-210 in canine neoplasms than in a control group was also observed in the study of Bulkowska et al. [113]. In HBC tissue, overexpression of miR-210 correlates with lymph node metastasis, clinical staging, differentiation and poor prognosis in patients with breast cancer. Therefore, miR-210 was proposed as a potential prognostic biomarker of breast cancer [159,160].

miR-138a
Among 18 significantly decreased miRNAs in the canine SNP cell line, miR-138a showed the greatest reduction in the expression (0.007-fold) [116]. As discussed in this study, tumor-suppressive miRNA-138a represses the epithelial-mesenchymal transition (EMT), a process resulting in cancer aggressiveness and metastasis. Since this study showed that some SNP cells were positive for vimentin as an important EMT marker [161], the authors declared that SNP cells undergo the EMT process, which also confirms the suppressive and biomarker role of miR-138a in CMC [116].

miR-8832, miR-96, and miR-149
Genome-wide methylation profiling in canine mammary tumors revealed miR-8832 as a new miRNA associated with both CMC and HBC [111]. Downregulated GNAO1 (guanine nucleotide-binding protein-alpha O1) in canine mammary tumors was predicted as its target gene. As discussed by the authors, this tumor suppressor gene is involved in the reduction of cell proliferation in some human cancers, and dysregulation of GNAO1 mRNA may be involved in tumorigenesis. Thus, miR-8832 represents a potential biomarker in both canines and humans [111].
The study also identified other miRNA candidates, upregulated miR-96 and downregulated miR-149, reported as cancer-associated miRNAs in humans [111]. Oncogenic miR-96 was found to be constantly upregulated in breast cancer tissues where it promotes proliferation, migration, and the invasion of cancer cells through silencing the target gene PTPN9 (gene for tyrosine-protein phosphatase non-receptor type 9) [162]. Tumor-suppressive miR-149 contributes to breast tumor progression by supporting aberrant Rac activation [163] and recruitment of macrophages to the tumor [164]. Using the sequence-based target prediction program TargetScan, the authors predicted BRPF3 (gene encoding a bromodomain and PHD finger containing 3), ADCY6 (gene encoding adenylyl cyclase type 6), and LRIG1 (gene encoding leucine-rich repeats and immunoglobulin-like domains protein 1) as targets for miR-96, and RNF2 (gene encoding E3 ubiquitin-protein ligase RING2) as a target for miR-149, highly conserved genes in dogs and humans [111].
Generally, miRNAs are more stable (up to 10-times) than mRNAs [165,166] and easy to detect in samples, such as tissues obtained from biopsy or surgery or biological fluids (such as serum, plasma, urine, saliva, seminal, ascites, amniotic pleural effusions, or cerebrospinal fluid) [167]. However, invasive procedures, such as tissue sample collection, are not very suitable for diagnostic or screening purposes, as mammary biopsies may yield a very small amount of RNA, with differences in quantified miRNAs at the level of one nucleotide [110]. In this regard, feasible and relatively non-invasive biofluid-extracted circulating miRNAs have attracted interest in the term of biomarkers as novel diagnostic tools for cancer, as this would limit the need for the collection of tissue samples and other invasive procedures [168]. Except for simple isolation, circulating miRNAs maintain stability under different conditions of sample processing and isolation [169]. Circulating miRNAs, as well as intracellular miRNAs, are also involved in the regulation of several biological processes with abnormal expression during pathological conditions [170]. Altered expression of circulating miRNAs is related to the initiation and progression of cancer [170]. Biofluid miRNAs show dynamic changes in physiological and pathological states before the clinical signs appear [171]. Furthermore, importantly, circulating miRNAs can be easily detected by basic molecular techniques [170]. Several circulating miRNAs have been described as biomarkers in cancer, including HBC (reviewed in [170,172]). Based on a literature review, we found four studies investigating levels of circulating miRNAs in plasma or serum samples in canine mammary tumors [113,[119][120][121]. Nonetheless, the first study by Bulkowska et al., comparing differences between metastatic and non-metastatic tumors, showed no significant differences in the expression of selected metastasis-specific miRNAs (cfa-miR-144, cfa-miR-32, cfa-miR-374a, and hsa-miR-1246) by polymerase chain reaction (PCR) analysis [113]. On the other hand, the recent study by Fish et al. revealed circulating miRNAs as biomarkers of canine mammary carcinoma [121]. In this work, serum miRNA from 10 healthy female dogs and 10 bitches with histologically confirmed mammary carcinoma revealed 452 unique serum miRNAs by RNA deep-sequencing and 65 miRNAs differentially expressed (>±1.5-fold) and statistically significant between groups (carcinoma vs. healthy) by digital droplet PCR (dPCR). Although the expression of several miRNAs, such as miR-29b, miR-34c, miR-122, miR-125a, and miR-181a, was found to be upregulated, the authors suggested differentially expressed circulating miR-18a and miR-19b as the most potential biomarkers.

Circulating miR-18a
Significantly upregulated serum miR-18a (1.94-fold by RNA sequencing; 1.24-fold by dPCR) was suggested as a candidate prognosis biomarker for CMC [121]. The authors revealed significantly higher levels of miR-18a in the group with histologic evidence of lymphatic metastasis invasion than without (2.82 versus 1.23 reads per million). Thus, miR-18a was proposed as a strong candidate prognostic biomarker also for HBC risk [121]. Circulating miR-18a was also overexpressed in a set of 60 serum samples from women with early-stage breast cancer compared to a sample of 51 healthy controls, suggesting miR-18a as a blood-based multi-marker for the early detection of HBC [173]. Generally, miR-18a, a member of the miR-17-92 cluster, suppresses the translation of estrogen receptor α (ERα), thus decreasing the protective effect of estrogen [174]. This finding was also observed in breast cancer-derived cell lines MCF-7 and MDA-MB-231, wherein not only the low expression of the ER, but also a decreased sensitivity to tamoxifen, and endocrine resistance, was associated with miR-18a high expression [175]. In another study, the overexpression of miR-18a in breast cancer cell lines MCF7 and ZR-75-1 led to an increase in the cells' proliferation and migration, significant repression of E-cadherin, activation of genes of the Wnt (Wingless and Int-1) noncanonical pathway, PCP (planar cell polarity) pathway, JNK (c-Jun N-terminal Kinase) pathway, and actin remodeling [176]. Furthermore, miR-18a was suggested as an early driver of tumorigenesis, since it was found to be upregulated in contralateral unaffected breasts and benign biopsy samples before the development of breast cancer [177].

Circulating miR-19b
Another significantly upregulated (3.15-fold by RNA sequencing; 1.76-fold by dPCR) serum miR-19b was proposed as a candidate diagnostic biomarker [121]. The ability to distinguish between mammary tumor-bearing dogs and dogs without neoplasia based on miR-19b was also revealed in this study with the ROC-AUC (receiver operator characteristic-area under the curve) and sensitivity/specificity analysis (ROC-AUC = 0.978) [121]. The miR-19b is a key molecule for cancer development, as it was found to be an active participant in the pathogenesis of various types of cancer, including HBC [178,179]. In breast cancer studies, miR-19b has demonstrated tumor-promoting activities. The wound-healing assay and transwell invasion assay performed by Zhao et al. demonstrated that overexpressed miR-19b facilitated the migration and metastasis of breast cancer cells by downregulation of myosin regulatory light chain interacting protein (MYLIP) involved in the regulation of cell movement and migration [179]. In the same study, miR-19b promoted the downregulation of E-cadherin and upregulation of intercellular adhesion molecule 1 (ICAM-1), and Integrin β1 in vitro and in vivo, leading to the activation of downstream signaling pathways (the Ras-MAPK pathway and the PI3K/AKT pathway) and involved genes [179]. In another study, miR-19b was found in less invasive breast lines (MCF-7, T47D, and ZR-75-1 cells) as well as in invasive breast lines (MDA-MB-231 and BT-20 cells), wherein it regulated at a post-transcriptional level the expression of tissue factor, known as a regulator of tumor angiogenesis and metastasis [178]. Taking together the results of these studies, miR-19b serves as an oncomiR in the progression of breast cancer and could act as a biomarker.

Circulating miR-21 and miR-29b
The latest studies from 2021 investigated serum miRNA-based biomarkers, miR-21 and miR-29b. Both miRNAs were also altered in tumor samples, as discussed above. In the study of Jain et al., serum samples of 60 female dogs (20 healthy/control, 20 with benign tumors, and 20 with malignant mammary tumors) were used [119]. Serum miR-21 was upregulated in malignant (3.0-fold) and benign (1.8-fold) tumors compared to the control samples (1.1-fold), while the expression of serum miR-29b was significantly downregulated in the malignant and benign group compared to the control samples (0.2-fold, 0.4-fold, and 1.1-fold, respectively). Interestingly, the expression was higher/lower in malignant tumors than in benign tumors. As suggested by the authors, circulating miR-21 could serve as a prognostic marker for the early detection of canine mammary tumors, and miR-29b can add sensitivity and accuracy to a diagnosis if evaluated together with miR-21 [119]. In the study of Ramadan et al., miR-21 was significantly upregulated (12.84-fold) in the serum samples of 10 female dogs with mammary tumors compared to the control group of 7 healthy bitches. Thus, miR-21 was hypothesized as a more sensitive, non-invasive indicator for CMC [120]. These observations are in accordance with other studies on tumor samples [110,113,114].
Despite the above-mentioned advantages of circulating miRNAs as biomarkers (nonor minimally invasive availability and easy accessibility, stability or resistance toward severe stressing conditions, such as high temperatures, repeated freeze-thaw cycles), they still have several issues hindering their reliability for the clinical application [180]. One of the major limitations of circulating miRNAs as biomarkers is the inability to identify their exact origin [181]. For example, most circulating miRNAs are obtained from blood using plasma or serum as the source [181,182]. However, blood contains a variety of cell types that challenge the identification of the cell origin of a particular miRNA [181]. The majority of the miRNAs in the blood are packaged in EVs like microvesicles (or m/lEVs) and exosomes (or sEVs) [180]. Exosomes and exosome-derived miRNAs have attracted great attention in recent years in terms of biomarkers [183]. A literature review of miRNAs from exosomal and non-exosomal sources showed that 71% of the selected articles concluded that exosomes are the source of choice for miRNAs in biomarker studies. In addition, 75% of articles comparing both sources of miRNAs recommended exosome-derived miRNAs over non-exosomal miRNAs [181]. Thus, it can be assumed that exosomes can be a better source of miRNAs as biomarkers due to their benefits in terms of quantity, quality, and stability [181], as discussed below.

Nomenclature
The International Society for Extracellular Vesicles (ISEV) approves the definition of EVs as lipid bilayer-surrounded particles released from the cell without the ability to replicate. Due to intersecting characteristics and the lack of consensus on specific markers of different EV subtypes (e.g., expression of CD9, probable marker of exosomes and ectosomes; [184]), some authors suggested rather to consider the origin of EVs. Based on this, the term exosomes should refer to the intracellular compartment-originated EVs and ectosomes (microparticles/microvesicles) as EVs derived from the plasma membrane [185,186]. However, the EVs' designation to a particular biogenesis pathway is challenging. Therefore, the ISEV proposed in 2018 "Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines" as recommendations for EVs nomenclature [90]. In total, 94% of MISEV2018 respondents affirm the classification of EVs subtypes according to either (i) physical characteristics such as size ("small EVs"; sEVs (<100 nm or <200 nm) and "medium/large EVs"; m/lEVs (>200 nm)) or density (low, middle, high), (ii) biochemical composition of surface markers (e.g., CD63+ EVs, CD81+ EVs, CD81− EVs, CD9+ EVs) or (iii) origin of parental cell or biological processes (e.g., tolerosomes, oncosomes, apoptotic bodies) [90]. However, the reviewed literature does not take into account the MISEV2018 guidelines and keeps the term "exosomes". To avoid misunderstanding in this review we decided to keep both terms "exosomes" and "sEVs".

Biogenesis
Since the identification of exosomes in sheep reticulocytes in the 1980s [187,188], these small endosomal-derived membrane vesicles have gained high interest over the last decade. sEVs (exosomes) are a subset of EVs secreted into the extracellular space by prokaryotic and eukaryotic cells, as well as in physiological and pathological processes [189]. To distinguish them from other EVs excluded from the body fluids, Rose Johnstone and colleagues gave them the name exosomes [190], now called sEVs based on the MISEV2018 guidelines [90]. As was described above, EVs are generally categorized based on their size into sEVs or m/lEVs [90]. Microvesicles (also known as ectosomes, microparticles or m/lEVs) have typically a diameter of medium/large-sized EVs (>200 nm-1000 nm) and are formed in the process of outward plasma membrane budding [90,96,97]. The suggested protein markers are CD40, selectins, and integrins [191]. Whereas sEVs (exosomes) and m/lEVs (microvesicles) are secreted during normal cellular processes, apoptotic bodies (>1000 nm) are only formed and released from the cells undergoing programmed death by plasma membrane blebbing [98,99] and express phosphatidylserine, the so-called "find-me, eat me" signal that triggers macrophage clearance [192][193][194]. Apoptotic bodies differ from the other two major EV groups by containing fragments of host DNA and cellular organelles [193]. These EVs can be distinguished by protein markers, such as histones, thrombospondins, and C3b [195]. sEVs (exosomes) are nano-sized (<200 nm) EVs surrounded by a lipid bilayer membrane which is characteristic for all EVs and protects the encapsulated material, such as nucleic acids (DNA, mRNA, and non-coding RNAs), proteins, peptides, chaperons, lipids, metabolites, from the extracellular environment ( Figure 2) [191,196]. Other authors subclassify the sEVs (exosomes) based on the size into exomeres (35 nm), small exosomes (Exo-S) (60-80 nm), and large exosomes (Exo-L) (>90 nm) [197]. sEVs (exosomes) are produced within the cells by an endocytic pathway regulated by proteins and lipids in form of the multivesicular bodies (MVBs) and released to the intercellular space after fusion with the cell membrane [94]. Shortly, sEV (exosome) maturation begins with the formation of the early secretory endosome mediated by clathrin-or caveolin-dependent or independent invagination of the cell membrane, together with the accumulation of bioactive substances [91][92][93]. The budding of the inner membrane of early endosomes leads to the maturation of the MVBs [91][92][93]. During this process, some proteins are incorporated into the invaginating membrane, while the cytosolic components (such as nucleic acids, protein, chaperones, peptides, metabolites, and lipids) are enclosed inside (Figure 2) [198]. MVBs are late endosomes containing intraluminal vesicles. MVBs are of two destinies: (1) direction to the lysosome for degradation by enzymes in the lysosome lumen; or (2) fusion with the plasma membrane to release the content (i.e., intraluminal vesicles) into the intercellular space ( Figure 1) [94,95]. The factors determining the direction of MVBs are still poorly known [199]. However, it was found that secreted MVBs contain an important pool of cholesterol [200]. This observation raises the question of whether high levels of cholesterol may be the determining parameter of MVBs' destiny. Most of the released intraluminal vesicles are sEVs (exosomes). The biogenesis of MVBs, together with exosome formation and release, is mediated by endosomal sorting complexes required for transport (ESCRT) mechanism and other ESCRT-associated proteins (vesicle trafficking 1, VTA1; apoptosis-linked gene 2-interacting protein X, ALIX; tumor susceptibility gene 101 protein, TSG101; or vacuolar protein sorting-associated protein; VPS4) [201][202][203]. The ESCRT is complex machinery that comprises four different types of multiprotein sub-unit complexes, named ESCRT-0 to III. ESCRT-0 is responsible for the recognition and recruitment of ubiquitinated cargo to the endosomal membrane, ESCRT-I and II for the membrane budding, and ESCRT-III mediates vesicle separation from the plasma membrane [204]. Additionally, recent evidence has demonstrated the effect of ESCRT-independent pathways on exosome formation [205][206][207]. It can be assumed that exosome formation is controlled by factors in the cell and tissue microenvironment [97,[208][209][210]. On the one hand, the production of sEVs (exosomes) is cell-regulated, as needed [97]. On the other hand, cell stress factors (hypoxia, acidosis) [208,209] or stimulation by growth factors (epidermal growth factor) [210] were found to induce exosome production and exocytosis. Several protein markers, including tetraspanins (CD9, CD63, CD81, and CD82), ALIX, TSG101, flotillin, heat shock proteins (HSP70, HSC70, HSP90), and T-complex protein 1 subunit beta (CCT2), are suggested as markers to differentiate sEVs (exosomes) from other EVs [211,212], even though they are recognizable by electron microscopy thanks to their typical biconcave or cup-like shape.
To allow the application of sEVs (exosomes) as biomarkers, effective isolation methods and optimal storage conditions are crucial. The most commonly used method is ultracentrifugation, followed by ultrafiltration, differential centrifugation, microfluid-based techniques, immunoaffinity chromatography, the polyethylene glycol-based precipitation method, and size-exclusion chromatography [260]. Each technique has its pros and cons and differs in the processing of the sample and the purity and quality of the exosomes obtained (reviewed in [261,262]  and several studies have demonstrated that different kits may introduce variations in the concentration, purity, and size of sEVs (exosomes) [263][264][265]. Thus, when evaluating results, it is necessary to take into account the advantages and disadvantages of individual isolation methods of EVs.
The great advantage of sEVs (exosomes) is the possibility of their long-term storage at lower temperatures before analysis, with no or minor impact on exosome yield or bioactivity [171,266]. However, storage temperature depends on the sEVs (exosomes) source. For example, urine exosomes are sensitive to the storage temperature [267]. Zhou et al. showed that storage of urine samples at −20 • C led to a significant loss of exosomes compared to freshly collected urine. Preservation at −80 • C combined with extensive vortexing after thawing maximized the efficiency of exosome recovery [267]. On the other hand, multiple studies have shown that blood components, such as plasma or serum, can be stored long-term (for several years) either at 4 • C, −20 • C or −80 • C, and even at room temperature for short time (1-2 days), with no significant exosome or exosome-associated RNA and proteins degradation [268][269][270][271][272]. However, the study of Dutta et al. showed a decrease in central nervous system-derived α-synuclein stability upon storing serum or plasma-originated exosomes after 5 years at −80 • C [273].
To summarize, the ability of exosomes to transfer regulatory messages to other cells and their availability and stability make them a valuable source of biomarkers.

Exosome-Derived miRNAs as Biomarkers
Nowadays, sEVs (exosomes) are of interest in biomarker research. Naturally, this raises the question of why exactly sEVs (exosomes)? Exosome cargo (represented by nucleic acids, proteins, peptides, lipids, and metabolites; Figure 2) is specific and may vastly differ among various cell types, even from the same primary cell [274], depending on their function and current state (e.g., normal, transformed, differentiated, stimulated, and stressed). Thus, cellor condition-specific sEV (exosome) content is something like a fingerprint of the donor cell reflecting the cellular processes and, therefore, may serve as biomarkers for various diseases [213]. Principally, the demonstration of miRNAs association with EVs by Valadi et al. in 2007 [71] open the way for a multitude of studies dealing with EV-associated miRNAs. Exosome-derived miRNAs have attracted considerable attention as non-invasive biomarkers of various diseases with diagnostic and prognostic potential [183,275,276]. To describe selectively packaged, secreted, and transferred miRNAs between cells in sEVs (exosomes) and distinguish them from circulating miRNAs, Bhome et al. introduced the term "exomiRs" [277]. These exomiRs offer some beneficial factors over circulating miRNAs that increased their importance as biomarkers. Except for the above-mentioned fact that the miRNA profile presents a signature of the parental cell, sEVs (exosomes)packaged miRNAs are highly protected from degradation, even in non-optimal storage conditions and in the presence of RNases, hence conditions that normally degrade free miRNAs [277][278][279]. Indeed, sEVs (exosomes) are considered to be a stable source of miRNAs, and exosomal miRNAs in biofluids are more stable in comparison to circulating miRNAs [280]. ExomiRs have been shown to maintain stability either for short-term storage (2 weeks) at 4 • C or long-term storage (5 years) at −20 • C, as well as resistance to freeze-thaw cycles [171]. Due to their ease of access and stability, exomiRs represent a minimally invasive tool for the diagnosis and prognosis of cancer. The fact that exomiRs are also secreted by other cell types and not only cancer cells could mask cancer-specific biomarkers [278]. Profiling multiple exomiRs markers and isolating exosomes using tumorspecific protein markers could improve exosomal miRNAs sensitivity and specificity [278].
Today, research generally monitors and measures miRNAs, as well as exomiRs, using microarrays and real-time PCR (RT-PCR) [275]. Microarrays can detect many aberrant miRNAs with the entire genome expression profiling of miRNAs in the sample, but without determination of absolute quantification [275,281]. Being more sensitive and specific, RT-PCR allows the detection of low-level miRNAs with the determination of absolute quantification [275,281]. However, it cannot be used to identify novel miRNAs [281]. Novel miRNAs and miRNAs distinguished only by one nucleotide can be detected by the accurate and sensitive method of RNA sequencing because no primers or probes are needed [275,281]. RNA sequencing was already applied in the detection of exosomal miRNAs [282][283][284].
To the best of our knowledge, in 2018, Fish et al. published the first and so far only study reporting the shedding of exosome-derived miRNA by canine mammary cells in vitro [309]. In particular, cell-free conditioned media containing exosome-like vesicles from three normal canine mammary epithelial cell cultures from canine patients without mammary pathology and five canine mammary tumor cell lines with histopathologyconfirmed mammary carcinoma (CMT12, CMT27, CMT28, CMT47, CMT119) were used to yield a number of significantly upregulated and downregulated exomiRs that may represent putative biomarkers of mammary neoplasia. This complex study detected 338 unique exomiRs with 145 differentially expressed exomiRs (118 upregulated and 27 downregulated) having >±1.5-fold difference between tumor and normal samples. Two proposed circulating low-invasive biomarkers in canine neoplastic diseases, including mammary carcinoma [315], miR-126, and miR-214, were also monitored in mammary tumors-exosomes. Generally, both miRNAs demonstrated a broad influence on cancer pathogenesis through the regulation of angiogenesis, proliferation, migration, and cancer cell death [316,317]. Therefore, alteration in their expression has a critical impact on tumor progression. In this study, miR-126 was found to be upregulated (2.25-fold). Thus, miR-126 may represent a prospective exosomal miRNA-based biomarker in canine mammary tumors. However, the expression of miR-214 was strongly downregulated (−9.13-fold) in the exosomal RNA of canine mammary tumors. As explained by the authors, high levels of miR-214 monitored in canine neoplastic diseases, including mammary cancer, can be either a result of secretion of other than canine mammary tumors cells (i.e., cells of the immune system, stroma or other organs) or a mismatch between tumor cell, exosomal and circulating miRNA profiles [309].
The findings of this study correlate with previously published studies on miRNAs in CMC discussed above. Several miRNAs, including miR-18a, miR-19a, miR-29b/c, miR-181a/b, miR-215, miR-345, miR-371, and miR-1841, were found to be upregulated in both canine mammary tumor cells and their exosomes [110,116,117,121]. However, some discrepancies in exomiRs expression levels compared to miRNAs profiles of tumor cells in other studies were observed, such as miR-19a, miR-29b/c, miR-31, miR-34c, miR-181a/b, miR-155, and miR-495 [113,114,117]. As discussed by the authors, this inconsistency may be a result of the active selection or enrichment process of particular miRNAs within exosomes or as a consequence of dramatic changes in tumor cell phenotype and gene expression in metastatic lesions [309].
In the same study, gene ontology enrichment analysis showed the cellular role of exomiRs in the regulation of enriched biological processes, such as positive regulation of cell proliferation, positive regulation of the apoptotic process, cell migration, response to hypoxia, regulation of gene expression, negative regulation of cell migration, or chromatin remodeling (histone ubiquitination or trimethylation) [309]. Target gene representation analysis associated with enriched gene ontology terms in order to select suitable candidates for clinical biomarker applications identified three miRNAs: miR-18a, miR-19a, and miR-181a [309]. These miRNAs were also the most significantly upregulated among all exomiRs (10.34-fold, 3.84-fold, and 7.70-fold, respectively) [309]. Moreover, miR-18a, miR-19a, and miR-181a were predicted in silico to target the estrogen receptor (ESR1α), the expression of which is known to be lost in human and canine neoplasms along with increasing grade and stage (miR-18a: miRDB target score = 99; miR-19a: miRDB target score = 71, and miR-181a: miRDB target score = 79) [309]. Based on these findings, the authors assume that miR-18a, miR-19a, and miR-181a represent non-invasive markers of hormone status and phenotype in CMC [309].

Conclusions
The non-exosomal and exosome-derived miRNAs identified in CMC as promising biomarkers reviewed in this study reveal heterogeneity in relation to expression level, potential use, and sampling (Table 2). Exosome-derived miRNAs miR-126 up diagnostic conditioned medium [309] miR-214 down diagnostic conditioned medium [309] miR-18a up diagnostic conditioned medium [309] miR-19a up diagnostic conditioned medium [309] miR-181a up diagnostic conditioned medium [309] Please note that miRNAs are listed based on their occurrence in the article.
However, CMC mimics human breast tumors in many aspects (histopathology, clinical outcome or molecular markers). The similarities in terms of function and dynamics of miRNAs in mammary/breast cancer point to the role of these small non-coding RNAs in Life 2022, 12, 524 20 of 34 cancer mechanisms of both canine and human origin. MiRNAs are post-transcriptional regulators of gene expression with an impact on practically all cellular physiological and pathological processes. Since miRNAs are involved in cancer-related processes (such as carcinogenesis, cell proliferation, invasion, metastasis, apoptosis or chemoresistance), their diagnostic, prognostic, and therapeutic significance has been proposed. Over the past few years, several studies regarding miRNA-based biomarkers of mammary cancer have been carried out in canine patients. Most of these studies were focused on miRNAs derived from tumors or cancer cell lines. However, a traditional solid biopsy is gradually receding and more often is being replaced by liquid biopsy, wherein biofluid-extracted biomarkers provide a platform for non-invasive or minimally invasive diagnosis and prognosis. sEVs (exosomes) are present in many biological fluids and can be used similarly for minimally invasive liquid biopsies in veterinary medicine. Furthermore, their cargo plays an important role in various physiological and pathological processes. In particular, exosome-derived miRNAs have been shown to have a complex role in tumorigenesis and tumor progression. However, utilizing sEVs (exosomes) and their exomiRs cargo as a diagnostic tool for CMC is still in its infancy and requires further investigation. Moreover, most of the presented studies were conducted on small groups of patients. Although all of the above-mentioned miRNAs-based biomarkers seem to have diagnostic or prognostic potential in CMC, more detailed studies should be carried out in the near future.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.