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Exosome proteomes reveal glycolysis-related enzyme enrichment in primary canine mammary gland tumor compared to metastases



Numerous evidence has highlighted the differences between primary tumors and metastases. Nonetheless, the differences in exosomal proteins derived from primary tumor and metastases remain elusive. Here, we aimed to identify differentially expressed exosomal proteins from primary canine mammary gland tumor and metastases to understand how they shape their own tumor microenvironment.


We clearly distinguished primary canine mammary gland tumors (CHMp) from metastases (CHMm) and profiled the proteins within their secreted exosomes using LC–MS/MS. Moreover, the abundance of glycolysis enzymes (GPI, LDHA) in CHMp exosome was verified with Western blotting, To broaden the scope, we extended to human colorectal cancer-derived exosomes (SW480 vs. SW620) for comparison.


We identified significant differences in 87 and 65 proteins derived from CHMp and CHMm, respectively. Notably, glycolysis enzymes (GPI, LDHA, LDHB, TPI1, and ALDOA) showed specific enrichment in exosomes from the primary tumor.


We observed significant differences in the cellular proteome between primary tumors and metastases, and intriguingly, we identified a parallel heterogeneity the protein composition of exosomes. Specifically, we reported that glycolysis enzymes were significantly enriched in CHMp exosomes compared to CHMm exosomes. We further demonstrated that this quantitative difference in glycolysis enzymes persisted across primary and metastases, extending to human colorectal cancer-derived exosomes (SW480 vs. SW620). Our findings of the specific enrichment of glycolysis enzymes in primary tumor-derived exosomes contribute to a better understanding of tumor microenvironment modulation and heterogeneity between primary tumors and metastases.


Ninety percent of cancer-related deaths are attributed to metastases [1]. To address this high mortality rate, understanding the distinctions between primary tumor and metastases is a crucial task in unravelling the intricacies of disease progression. The primary tumor is where cancer originates and initially manifests, representing the early genetic changes and molecular characteristics that determine its origin [2]. However, an intriguing aspect is that, even if they share the same origin, primary tumors and metastases have distinct cellular characteristics [3]. Based on these disparities, primary tumors and metastases have different communication systems in forming their tumor microenvironment. Accumulating evidence revealed that cancer cells not only directly interact with surrounding cells such as fibroblasts, endothelial cells, and immune cells within the tumor microenvironment but also indirectly create their desired niche by secreting soluble factors [4, 5]. The communication between cancer cells and the surrounding microenvironment is complex and involves various signaling pathways and interactions [6,7,8]. Through this intricate interplay, cancer cells can modulate the tumor microenvironment to support their survival, growth, invasion, and the formation of metastases [9]. Recently, the role of signaling molecules in regulating the tumor niche has been well established not only for soluble proteins but also for exosomes [10, 11].

Exosomes, a specific type of extracellular vesicle, are secreted by various cells and typically range in size from 30 to 150 nm are found in biological fluids [12]. It is known that cancer cells secrete a higher number of exosomes compared to normal cells. These exosomes contain proteins, RNAs, DNAs, and non-coding RNAs [13, 14]. The components within exosomes are shuttled through various mechanisms within cells, reflecting the characteristics of the parent cells [15]. The composition of exosomes can vary depending on the cell type and the conditions under which they are secreted [16]. Numerous studies have shown that exosomes derived from cancer cells carry oncogenic proteins, mRNAs, and ncRNAs, which can contribute to the formation of a metastatic niche in nearby or distant cells [17,18,19]. However, many studies tend to overlook the distinct heterogeneity between primary tumors and metastases and treat them without clear distinction.

In this study, our objective was to conduct a detailed profiling and identification of distinct proteins present in exosomes derived from primary tumors compared to those originating from metastases. We used canine mammary gland tumor patients-derived cell lines; CHMp for primary tumor and CHMm for metastases. Through the utilization of LC–MS/MS, we conducted an extensive analysis to uncover unique protein signatures inherent to exosomes derived from primary tumors and metastases. Proteome analysis of these exosomes revealed significant differences between them. More importantly, glycolysis enzymes (GPI, LDHA, TPI1, and ALDOA) were significantly enriched in the exosomes of primary tumors of both canine mammary tumor and human colorectal tumor, compared to metastases.


Cell culture

Canine mammary tumor cells (CHMp and CHMm) were established and obtained from the N. Sasaki lab [20]. CHMp, derived from primary tumors and CHMm, derived from metastatic cancer, were maintained in RPM I1640 medium (Hyclone, SH30027) supplemented with 10% fetal bovine serum (FBS; Gibco, 1,600,044) and 50 ug/ml gentamicin (Sigma‒Aldrich, G1272) at 37 °C humidified incubator with 5% CO2. Two cell lines used in the study were authenticated, Mycoplasma-free.

Exosome isolation

Exosome isolation was performed as previously described [21], and the isolation method is summarized in Fig. 1A. Briefly, CHMp and CHMm cells were cultured until reaching 80–90% confluency. Twenty-four hours before exosome isolation, cells were washed twice with PBS and then cultured with serum-free RPMI medium without any supplements. The culture supernatant (CS) was collected and subjected to differential centrifugation: 300 × g for 10 min to remove dead cells, 2,000 × g for 10 min to remove cell debris and 10,000 × g for 30 min. The supernatant was further centrifuged at 100,000 × g for 80 min. The pellet from ultracentrifuge was washed once with PBS and resuspended with appropriate buffers for the assay. All centrifugation steps were performed at 4 °C.

Fig. 1
figure 1

Isolation and characterization of exosomes. A Schematic flow of exosome isolation. B Transmission electron microscopy of exosomes from CHMp and CHMm cells. Exosomes are negative stained. Isolated exosomes show a cup-shaped morphology. Scale bar: 200 nm. C Dynamic light scattering analysis of CHMp and CHMm-derived exosomes. The mean diameter size of CHMp-derived exosomes were 91.6 ± 17.5 nm and CHMm-derived exosomes were 33.0 ± 0.0 nm

Electron microscopy (EM)

Isolated exosomes were dissolved in PBS at a concentration of 1 μg/μL. Subsequently, 1 μg of exosomes was placed on glow discharged carbon-coated copper grids for 1 min. Excess liquid on the grid was removed using filter paper, and negative staining was performed using a 2% (v/v) uranyl acetate solution for 10 min. After draining the staining solution, the grids were air-dried, and they were immediately observed under transmission electron microscopy (TEM) at 120 kV. TEM imaging was conducted using a TEM Talos L120C (Czech) which located at the NICEM at the Seoul National University.

Western blot assay

The Western blot assay was performed following a previously reported method [22]. Briefly, exosomes were lysed using RIPA buffer supplemented with 4% (v/v) 25 × protease (Roche, 04693116001) and 10% (v/v) phosphatase inhibitor cocktails (Roche, 049068545001). The lysed exosomal proteins were quantified using a BCA assay, and equal concentrations of CHMp, CHMm SW480, and SW620 exosomal proteins were loaded onto SDS-PAGE gels. The proteins separated by SDS-PAGE were transferred to an 0.2 µm nitrocellulose membrane (Amersham™ Protan™, 10,600,004) followed by overnight incubation with respective primary antibodies at 4 °C. Subsequently, after three washes with 0.05% (v/v) TBS/Tween 20, secondary antibodies were applied. After three washes with 0.05% (v/v) TBS/Tween 20, membrane was subjected to chemiluminescence detection using ECL (Biomax, BWP0200). (Primary antibodies; LDHA (expected molecular weight; 37 kDa), Cell Signaling Technology, 3582 T, 1:1,000, and GPI (expected molecular weight; 60 kDa), Cell signaling Technology 94,068, 1:1,000, Secondary antibodies; Goat anti-Rabbit IgG + H + I HRP conjugated, Bethyl, A90-116P, 1:3,000).

Proteomics sampling and LC–MS/MS

Exosomal proteins from CHMp and CHMm (50 µg each) were digested with trypsin according to filter-aided sample preparation (FASP) digestion method [23]. FASP digestion was performed as previously reported in our laboratory [24]. Briefly, 50 µg of protein were mixed with 200ul of 8 M urea in 30 K Microcon devices (Millipore, YM-3). The reduction (10 mM of TCEP, Tris (2-carboxyethyl) phosphine) and alkylation (40 mM of IAA, iodoacetamide) of proteins were performed on 30 K Microcon with centrifugation washing. The resulting concentrates were digested with Pierce MS-grade trypsin for overnight at 37’C and desalted using StageTip C18 method. Briefly, The C18 stage tip was made by mounting three C18 discs (Empore, 2215) for reversed-phase material. Each StageTip was activated with sequentially 100% methanol, 80% (v/v) acetonitrile (ACN) in 0.1% formic acid and 0.1% formic acid. Next, peptides were loaded and washed with 0.1% formic acid. Finally, elution of peptides was performed using 60% (v/v) ACN in HLPC-grade water. Eluted peptides were quantified by BCA peptide assay (Thermo Fisher Scientific, 23,275) and 25 µg of peptides labeled with Tandem Mass tag (TMT) six-plex isobaric label reagent (Thermo Fisher Scientific, 90,061) following the manufacturer’s recommendation. The labeled peptides were fractionated into three parts using by SDB-RPS (poly(styrenedivinylbenzene) reverse phase sulfonate). The detailed protocol and buffer compositions were described at Mann et al. [25].

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis was performed with a Q Exactive (Thermo Fisher Scientific) coupled with an EASY-nLC1200 UHPLC system (Thermo Fisher Scientific) as previously reported in our lab [22]. The peptides were injected into an EASY-Spray column (75 μm i.d. × 50 cm; PepMap RSLC C18 particle, 2 μm particle size, 100 Å pore size) and subjected to a 90-min LC gradient at a flow rate 250 nl−1. The MS data were acquired in data-dependent mode and the full scan resolution was set to 120,000 at m/z 400. MS/MS raw data were processed with MaxQuant (ver1.6.5.1) software and Uniprot dog proteome (number of entries: 43,621) was used for database searching. Proteins identified with two or more unique peptides (> 9 amino acids) were considered significant. The false-discovery rates (FDRs) were less than 1% at global protein level. TMT intensity efficiency, multi-scatter plots and Principal Component Analysis (PCA) of identified exosomal proteins were analyzed using Perseus software (v1.6.15.0).

Bioinformatic analysis

The UniProt database was employed to categorize protein based on their subcellular localization. Gene Ontology (GO) analysis was carried out using the DAVID functional annotation tool (v6.8) ( For network analysis, the STRING DB (v11) ( was utilized. Additionally, Gene set enrichment analysis (GSEA) plots of differentially enriched proteins were generated using GSEA software (v.4.1.0).


Isolation and characterization of cancer cell-derived exosomes

To investigate differences between primary tumor and metastases, we aimed to use tumor cell lines originated from same individual patient. Unfortunately, no paired human breast cancer cell lines were available for this purpose. Thus we choose to utilize canine mammary gland tumor cell lines; CHMp representing the primary tumor and CHMm representing metastases, derived from metastatic lung pleural effusion originated from same individual [20]. Canine mammary gland tumor cell lines, CHMp and CHMm was driven by their natural occurrence and the gene regulatory sequence and reference genome of dogs is more similar to humans than mice [21, 26].

We previously established an exosome isolation method to purify exosomes from CHMp and CHMm [21]. The detailed strategy is outlined in Fig. 1A. To further characterize the morphology of CHMp and CHMm-derived exosomes, negative staining was performed, and transmission electron microscope (TEM) was used. The isolated exosomes exhibited cup-shaped membranous vesicles with sizes below 200 nm, and there was no significant difference in the morphology of exosomes between the CHMp and CHMm (Fig. 1B). Exosome diameter measurement by dynamic light scattering (DLS) showed that the average diameter of exosomes derived from CHMp was within 91.6 ± 17.5 nm, while exosomes derived from CHMm were approximately 33.0 ± 0.0 nm in size (Fig. 1C).

Proteomic profiling and comparison between CHMp- and CHMm-derived exosomes

Next, we analyzed the proteins within the cell-derived exosomes. The proteins isolated from the exosomes were digested into peptides using the FASP method. Technical replicates consisting of three samples each from CHMp and CHMm, were labeled with TMT, fractionated, and subjected to LC–MS/MS (Fig. 2A). The exosomal proteins derived from CHMp and CHMm were analyzed in biological triplicates, and the TMT intensity for each replicate was uniformly labeled across all replicates (Fig. 2B). To assess the correlation among replicates for the CHMp and CHMm exosomal proteins, we performed principal component (PC) analysis which demonstrated a high correlation (> 0.9) between replicates (Fig. 2C,D). The exosome markers CD9, CD63, CD82, TSG101, Alix and GAPDH were enriched in all exosomes, while other markers such as Nucleus (HSP90B1, HIST2H3A, SUB1), Golgi (MAN2A1, BTGALT1), Lysosome (LAMP1), and Mitochondria (ITGB5) were not (Fig. 2E). Out of total 1,284 identified proteins, 87 proteins were enriched in CHMp exosomes by log2(fold change) > 1.2 and p-value < 0.05 and 65 proteins in CHMm exosomes by log2(fold change) < -1.2 and p-value < 0.05 (Fig. 2F, and Table 1, 2, 3 and 4). The 87 CHMp exosomal proteins were predominantly localized in the extracellular region or nucleus, while 65 CHMm exosomal proteins showed different localization patterns, mainly in the extracellular region or plasma membrane (Fig. 2G). These results suggest significant differences in the composition of exosomal proteins derived from primary tumors and metastases, indicating that heterogeneity between primary tumors and metastases is reflected in the composition of exosomal proteins.

Fig. 2
figure 2

Proteomic profile of CHMp and CHMm-derived exosomes. A Schematic figure of exosome proteomics analysis. B TMT labeling intensity efficiency between CHMp and CHMm exosomal proteins in triplicates. C-D Principal Component Analysis (PCA) of CHMp and CHMm exosomal proteome replicates. C TMT intensity correlation of the replicates of CHMp and CHMm exosomal proteins. PCA showed high correlation for technical and biological replicates. D PCA showed high correlations of CHMp and CHMm exosomal protein triplicates. The X and Y axes show principal component 1 and principal component 2, respectively. E Heatmap of 13 exosomal proteins representing cellular localization. CD9, CD63, CD82, TSG101, Alix and GAPDH for exosome, HSP90B1, HIST2H3A and SUB1 for nucleus, MAN2A1, BTGALT1 for golgi, LAMP1 for lysosome, ITGB5 for mitochondria. F Volcano plot based on the Log2 (fold change) and their -Log10 (p-value) of CHMp and CHMm exosomal proteins. In the plot, red dots indicate the proteins that are statistically enriched in CHMp exosomes, blue dots represent proteins enriched in CHMm exosomes, and grey dots represent proteins that are not statistically significant. Significantly enriched proteins in CHMp exosomes compared with CHMm exosomes as control, Student’s t-test, p < 0.05, obtained in Perseus software. G The subcellular localization of CHMp and CHMm exosomal proteins showing significantly differential expressions. Predicted subcellular localization were obtained from UniProt ( CHMp exosomal proteins were predominantly located in the nucleus and extracellular region. n = 3 biologically independent exosomal protein isolations. Figure 2A created with

Table 1 Total protein list
Table 2 CHMp enriched exosomal protein list
Table 3 CHMm enriched exosomal protein list
Table 4 Differentially expressed top 10 proteins in CHMp and CHMm-derived exosome

Protein interactions identified in CHMp and CHMm exosomal proteins

To gain a better understanding of the function of the identified exosomal proteins, we conducted Gene ontology (GO), STRING (Search Tool for the Retrieval of Interacting Genes) and Gene Set Enrichment Analysis (GSEA) (Fig. 3). GO analysis was conducted for each group, including biological process (BP), cellular component (CC), and molecular function (MF) (Fig. 3A). CHMp exosomal proteins were significantly enriched in Collagen, Poly (A) RNA, and protein bindings, whereas CHMm exosomal proteins were mainly involved in the extracellular matrix proteins organization and binding (Integrin, Laminin, and Cadherin). The protein interaction hubs of the CHMp and CHMm exosomal proteins were found to be completely different. The terms of "Proteasome", "Glycolysis/Gluconeogenesis", “Splicing factor” and "Extracellular matrix-collagen" were exclusively composed of CHMp exosomal proteins (Fig. 3B). On the other hand, interactions with laminin (LAMA3, LAMA5, LAMB1, LAMB3, LAMC1, and LAMC2) proteins were observed in CHMm exosomal proteins (Fig. 3C). These protein–protein interaction (PPI) hubs were also reflected in the Gene Set Enrichment Analysis (GSEA). The GO analysis of CHMp exosomal proteins revealed enrichment in the "carbohydrate catabolic process", while the Reactome analysis showed enrichment in "TCR signaling" (Fig. 3C). In addition, the GO analysis of CHMm exosomal proteins showed enrichment in the "positive regulation of GTPase activity", and the Reactome analysis indicated enrichment in "laminin interaction" (Fig. 3D). These results indicate that the composition of exosomal proteins differs between the two cell types, leading to distinct predicted functions. This highlights the potential functional variations of these exosomes in relation to their tumor microenvironment and target cells.

Fig. 3
figure 3

Comparative proteomic analysis between CHMp and CHMm exosomal proteins. A Gene Ontology (GO) analysis of statistically enriched in CHMp and CHMm exosomal proteins by the web tool DAVID v 6.8. (Yellow bars; BP, biological process. Green bars; CC, cellular component, blue bars; MF, molecular function). B STRING network analysis of differentially expressed proteins (DEPs) in CHMp exosomal proteins. Protein–Protein Interaction (PPI) network showing that CHMP exosomal proteins are enriched in proteasome, glycolysis/gluconeogenesis, splicing factor and extracellular matrix-collagen. C STRING network analysis of DEPs in CHMm exosomal proteins. CHMm exosomal proteins are enriched in Laminin. Proteins are shown as nodes. D-E Gene set enrichment analysis (GSEA) between DEPs in CHMp and CHMm exosomal proteins. D GSEA revealed carbohydrate catabolic process were significantly enriched in CHMp exosomal proteins. E Laminin pathways enriched in CHMm exosomal proteins

Glycolysis enzymes were enriched in primary tumor derived exosomes

To investigate whether the differences observed in canine mammary gland tumor-derived exosomal proteins are applicable to other species and cancers, we conducted a comparative analysis using proteomic data from human primary (SW480) and metastatic (SW620) colorectal cancer-derived exosomes [27]. We selected SW480 enriched exosomal proteins with fold change (fold change) > 1.2 and p-value < 0.05 and compared them with CHMp exosomal proteins. Among the selected proteins, we found 19 proteins that were more abundant in both SW480 and CHMp exosomal proteins, with several proteins related to glycolysis/gluconeogenesis (GPI, LDHA, LDHB, TPI1, and ALDOA) being commonly enriched (Fig. 4A). Notably, GPI showed more than a three-fold enrichment in both the CHMp and SW480 primary tumor exosomes compared their respective metastases (Fig. 4B and Supporting Information Table 1). The glycolysis enzymes enriched in the exosomes of primary tumors are all involved in lactate production during the glycolysis process (Fig. 4C). Western blot showed that GPI proteins were significantly enriched in primary tumor-derived exosomes across species and cancers. LDHA proteins were enriched only in CHMp-derived exosomes (Fig. 4D). We confirmed the levels of Alix in exosomes to serve as a loading control. Among the genes of the five proteins selected, GPI, LDHA, TPI1, and ALDOA showed high expression in both breast cancer and colorectal cancer and were associated with poor patient prognosis (Fig. 4E). In summary, we identified glycolysis enzymes specifically enriched in the exosomes derived from primary tumors. These findings suggest that primary tumor-derived exosomes are likely to affect on the lactate production of neighboring tumor microenvironment or distant target cells and have implications for cancer prognosis.

Fig. 4
figure 4

Comparative analysis of primary tumors-derived exosomal proteins between CHMp and SW480. A Venn diagram illustrating the 19 common proteins between CHMp and SW480 DEPs. Venn diagram visualized using the Venny 2.1. DEPs satisfying the fold change > 1.2 and p-value < 0.05. B The five proteins commonly enriched in both CHMp and SW480 exosomal proteins. (Purple bars; CHMp enriched, yellow bars; SW480 enriched). GPI showed highly enriched in both CHMp and SW480. C Glycolysis and gluconeogenesis pathway. Primary tumor enriched enzymes (GPI, LDHA, LDHB, TPI1, and ALDOA) were bolded. D Western blot analysis of primary tumor and metastases-derived exosomes for GPI and LDHA. GPI showed enrichment both in CHMp and SW480, whereas LDHA showed enrichment only in CHMp exosomes. Alix was used as loading control. E Kaplan–Meier survival curves of GPI, LDHA, TPI1, and ALDOA of breast cancer and colorectal cancer patients. High-level expressions of GPI, LDHA, TPI1 and ALDOA in breast and colorectal cancer patients showed worse overall survival outcomes. Kaplan–Meier plots derived from


Metastases exhibit unique characteristics and high heterogeneity due to the differential tumor microenvironment (TME) in which they reside [3, 28]. To gain a deeper understanding of the heterogeneity between primary tumors and metastases, we focused on analyzing proteins respectively. In our study, we employed LC–MS/MS to screen exosomal proteins, providing insights into the molecular composition of exosomes derived from primary tumors and metastases. Notably, our analysis revealed a significant enrichment of glycolysis enzymes specifically in the exosomes derived from primary tumors. This finding suggests that primary tumor-derived exosomes may play a role in modulating the metabolic landscape of the tumor microenvironment.

Canine mammary tumors (CMT) are the most common disease in female dogs. Recently, the use of CMT as a model for human breast cancer has been recognized in the field of comparative medicine. This approach is justified by the genetic and epigenetic proximity between dogs and humans, surpassing that of mice. Furthermore, human breast cancer and CMT not only share the commonality of being spontaneous cancers but also exhibit similarities in epidemiological, environmental, and pathological features, including histological and molecular heterogeneity.

We conducted a comparative analysis using the exosome proteomics data from human primary and metastatic colorectal cancer cells (SW480 and SW620). This analysis also revealed a significant enrichment of glycolysis enzymes in primary tumor-derived exosomes, consistent with our findings in CHMp exosomes. Glycolysis, a key metabolic pathway involved in energy production and biosynthesis in cancer cells [24], is closely associated with tumor metastasis [25]. Exosome-mediated metabolic reprogramming plays a crucial role in tumor microenvironment formation and tumor progression [26], affecting various surrounding cell types, including normal fibroblasts, cancer-associated fibroblasts (CAFs), mesenchymal cells (MSCs), endothelial cells (ECs), and immune cells. The key mediators involved in this process are the miRNAs and proteins within the exosomes. Exosomal miRNAs such as miR-105, miR-155, and miR-210 derived from cancer cells have been shown to increase glycolysis in CAFs, leading to enhanced lactate production that fuels cancer cell growth. Among exosomal proteins, VEGF found in exosomes stimulates glycolysis in endothelial cells, while the glycolytic enzyme PKM2 enhances glycolysis in MSCs [26].

A recent report revealed that exosomes derived from cancer cell lines with high metastatic potential contain a greater abundance of glycolytic enzymes [29]. Notably, GPI showed substantial enrichment in both primary tumor-derived exosomes and primary colorectal cancer-derived exosomes compared to their respective metastatic counterparts. In various cancers, GPI expression is increased by c-Myc and HIF-1 [30]. GPI knock-out (KO) has been shown to inhibit cancer cell growth [31], suggesting a significant role for GPI in cancer progression. Understanding the mechanisms underlying exosomal transfer of GPI and its impact on the TME indicates the importance of communication between cancer cells and their microenvironment. Further research is warranted to elucidate the specific role of exosomal GPI in mediating cancer cell growth and its implications for tumor progression and therapeutic interventions targeting the TME.

Not only GPI but also these glycolysis enzymes, except for LDHB, have been associated with patient’s poor prognosis in breast cancer and colorectal cancer. These findings suggest that primary tumor-derived exosomes may influence lactate production in the tumor microenvironment or distant cells, thereby impacting cancer prognosis [32, 33]. Our findings also indicate that lactate production enzymes enriched in the exosomes are not limited to a specific species or type of cancer, but represent a characteristic of primary tumors independent of species or cancer types. Moreover, exploring the enrichment of glycolysis enzymes in the exosomes may uncover their potential role in shaping the metabolic microenvironment.

Similar to glycolysis enzymes, splicing factors specifically enriched in primary tumors compared to metastases have been reported to play a role in the Epithelial-Mesenchymal Transition (EMT) during the metastatic progression of cancer [34]. This suggests their significant involvement in tumor progression. Splicing factors play a crucial role in RNA splicing, a process that transforms the initial RNA transcript (pre-mRNA) generated by the transcriptional apparatus into mature mRNA. Recently, it has been revealed that splicing factors are involved in the regulation of the Epithelial-Mesenchymal Transition (EMT) in the metastatic cascade of cancer. Moreover, numerous core splicing complexes (e.g., SF3B1, SF3B2, and SFRS1) in oncogenic Madin-Darby canine kidney cell-derived exosomes have been identified to promote metastatic progression [35]. Additionally, it has been reported that splicing components within exosomes are involved in the selective enrichment of miRNA. Splicing factors (SRSF1, EIF3B, TIA1) are implicated in the enrichment of pancreatic cancer-derived exosomal miRNA, particularly contributing to the exosome shuttling of miR-1246 [36]. Thus, exosomal spliceosome components, by participating in the selective shuttling of exosomal miRNA, imply that the functions manifested in cells may vary depending on which miRNA is shuttled into exosomes by spliceosome components. Exosomal miRNA, plays a crucial role in the tumor microenvironment. Considering the imbalanced enrichment of spliceosome components, further study is needed to investigate the differences in miRNA within exosomes derived from primary tumors and metastases.

The role of the proteasome in cancer involves its crucial function in maintaining proteostasis within cells by removing short-lived regulatory proteins and damaged proteins. The eukaryotic 26S proteasome is composed of the 20S core particle proteasome and the 19S regulatory particle (RP). The 20S core protein consists of 7 alpha subunits and 7 beta subunits, with the alpha subunits forming a cylindrical structure. Notably, beta subunits, specifically beta 1, beta 2, and beta 5, possess hydrolytic activity, cleaving the C-terminal peptide bond behind specific amino acids to exhibit Thr protease activity [37]. Exosomes derived from primary cancer contain a higher abundance of proteasome subunits compared to those from metastatic cancer. Specifically, they significantly contain all alpha subunits except for alpha subunit 4, and beta subunits with catalytic activity, such as beta subunit 2, 5, 3, and 4. Recent proteomic analyses of exosomal proteins have revealed the presence of numerous proteasome subunits in exosomes [38]. In exosomes derived from a mouse model of prostate cancer, all subunits of the 20S proteasome were confirmed, and tumor-associated macrophages exhibited a higher abundance of proteasomes in exosomes compared to naïve macrophages [38]. Moreover, studies suggest that exosomes can induce angiogenesis and enhance metastatic activity. Additionally, in mice with vascular injury, an increase in apoptotic exosomes containing the 20S proteasome core has been reported, indicating potential implications in autoantibody production and rejection acceleration [39]. Although the presence of proteasomes in exosomes has been observed, their exact role within exosomes remains elusive. Whether exosomes serve to deliver proteasomes to other cells or discard intracellular proteasomes is an area yet to be fully understood. Further research is needed to unravel the specific purpose, origin, mechanisms, and substrate specificity of these 20S proteasomes for understanding the role of primary tumor-derived exosomes. In this study, we report the identification of proteasome subunits are unique to the CHMp exosomal protein.

Overall, unraveling the connections between primary tumors and glycolysis-related exosomal proteins can provide a deeper understanding of the mechanisms driving cancer progression and open new avenues for the development of targeted therapeutic strategies aimed at disrupting these processes.


In this study, through LC–MS/MS, we discovered significant differences between the CHMp and CHMm-derived exosomal proteins. Enrichment of glycolysis enzymes (GPI, LDHA, LDHB, TPI1, and ALDOA) in exosomes derived from primary tumors indicates their potential contribution to metabolic reprogramming on nearby or distant target cells. However, this study is limited by the use of only two cell lines, and the specific mechanisms by which enzymes involved in this process may impact tumor microenvironment cells have not been elucidated. In future studies, we will further expand our analysis by including different types of primary and metastatic cell lines for comparison and investigate the specific functions of these glycolysis enzymes while exploring their roles in tumorigenesis. In summary, larger and more in-depth studies are crucially needed to elucidate the roles of glycolysis enzymes derived from primary tumors.

Availability of data and materials

All data supporting the findings in this study can be obtained from the corresponding author upon reasonable request.



Liquid chromatography—mass spectrometry


Glucose phosphate isomerase


Lactate dehydrogenase A


Lactate dehydrogenase B


Triosephosphate Isomerase 1


Aldolase A


Tumor microenvironment


Filter-aided sample preparation


Poly(styrenedivinylbenzene) reverse phase sulfonate


Transmission electron microscope


Dynamic light scattering


Glyceraldehyde-3-phosphate dehydrogenase


Heat Shock Protein 90 Beta Family Member 1


Histone cluster 2 H3 family


SUB1 Regulator of Transcription


Mannosidase Alpha Class 2A Member 1


β-1, 4-Galactosyltransferase gene


Lysosomal Associated Membrane Protein 1


Integrin Subunit Beta


Protein-protein interaction


Search Tool for the Retrieval of Interacting Genes


Gene Set Enrichment Analysis


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We thank the Korea Basic Science Institute (KBSI) for their mass spectrometric measurements.


This research was supported by grants from the Ministry of Science and ICT and the National Research Foundation of Korea (NRF) SRC program: Comparative Medicine Disease Research Center (CDRC) (2021R1A5A1033157) and National Research Foundation of Korea (NRF) (2014M3A9D5A01073598).

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H.K designed and performed experiments, wrote the primary manuscript. J.-Y.C. conceived the idea, provided scientific direction, revised the manuscript, and supervised the entire study.

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Correspondence to Je-Yoel Cho.

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Kim, HS., Cho, JY. Exosome proteomes reveal glycolysis-related enzyme enrichment in primary canine mammary gland tumor compared to metastases. Proteome Sci 22, 4 (2024).

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