Exosome proteomes reveal glycolysis-related enzyme enrichment in primary tumors

doi:10.21203/rs.3.rs-3341790/v1

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Research Article

Exosome proteomes reveal glycolysis-related enzyme enrichment in primary tumors

https://doi.org/10.21203/rs.3.rs-3341790/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 28 Feb, 2024

Read the published version in Proteome Science →

You are reading this latest preprint version

Objective

In this study, we aimed to identify differentially expressed exosomal proteins from primary tumor and metastases.

Methods

We clearly distinguished primary 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) was verified with Western blotting, and we extended to human colorectal cancer-derived exosomes (SW480 vs. SW620) for comparison.

Results

Among all the exosomal proteins derived from primary tumor and metastases, 87 and 63 proteins, respectively, exhibited significant differences. Notably, glycolysis enzymes (GPI, LDHA, LDHB, TPI1, and ALDOA) were specifically enriched in exosomal proteins from primary tumor.

Conclusion

We reported significant differences in the proteome at the cellular level between primary tumors and metastases, and intriguingly, we found this heterogeneity was mirrored in the protein composition of exosomes. We discovered 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 metastatic cancers, 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.

Cancer

Primary tumor

Metastases

Exosome

Proteomics

Ninety percent of cancer-related deaths are attributed to metastases [1]. In breast cancer, 75.6% of patients die from metastatic disease [2]. Breast cancer ranks as the most commonly diagnosed cancer in women and holds the second position in terms of mortality rates. 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 [3, 4]. The communication between cancer cells and the surrounding microenvironment is complex and involves various signaling pathways and interactions [5–7]. Through this intricate interplay, cancer cells can modulate the tumor microenvironment to support their survival, growth, invasion, and the formation of metastases [8]. Recently, the role of signaling molecules in regulating the tumor niche has been well established not only for soluble proteins but also for exosomes [9, 10].

Exosomes, a specific type of extracellular vesicle, are secreted by various cells and typically range in size from 30 to 150nm can be found in biological fluids [11]. It is known that cancer cells secrete a higher number of exosomes compared to normal cells, and exosomes contain proteins, RNAs, DNAs, and non-coding RNAs [12, 13]. The components within exosomes are shuttled through various mechanisms within cells, and they have been found to reflect the characteristics of the parent cells [14]. The composition of exosomes can vary depending on the cell type and the conditions under which they are secreted [15]. 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 [16–18]. 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 in comparison to those originating from 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 maintained in RPM I1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS; Gibco) and 50 ug/ml gentamicin (Sigma‒Aldrich) at 37°C humidified incubator with 5% CO2.

Exosome isolation

Exosome isolation was performed as previously described [19]. Exosome isolation method was summarized in Fig. 1A. Briefly, CHMp and CHMm cells were cultured until cell density reached at 80–90% confluency. Before 24hr for exosome isolation, cells were washed with PBS for 2 times and then culture with serum free RPMI medium without any supplements. Culture supernatant (CS) was collected and differential centrifuged at 300 x g for 10 min, to remove dead cells, 2,000 x g for 10 min for remove cell debris and 10,000 x g for 30min. The supernatant was further centrifuged at 100,000 x g for 80 min. Pellet from ultracentrifuge was washed with PBS once and resuspend with appropriate buffers for the assay. All of centrifugation was performed at 4 ^oC.

Electron microscopy (EM)

Isolated exosomes were dissolved in PBS at a concentration of 1 µg/µL. A total of 1 µg of exosomes was placed on glow discharged carbon-coated copper grids for 1 minute. Excess liquid on the grid was removed using filter paper, and negative staining was performed using a 2% uranyl acetate solution. After draining the staining solution, the grids were air-dried, and TEM imaging was conducted immediately using an operating voltage of 120 kV.

Western blot assay

The Western blot assay was performed following a previously reported method [20]. Briefly, exosomes were lysed using RIPA buffer supplemented with protease and phosphatase inhibitor cocktails. The lysed exosomal proteins were quantified using a BCA assay, and equal concentrations of CHMp and CHMm exosomal proteins were loaded onto SDS-PAGE gels. (Primary antibodies ; LDHA, Cell Signaling Technology, 3582T, 1:1,000, and GPI, Cell signaling Technology 94068, 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 protein from CHMp and CHMm (50 µg each) were digested with trypsin according to filter-aided sample preparation (FASP) digestion [21]. Briefly, 50 µg of protein were mix with 200ul of 8M urea in 30K Microcon devices (YM-3, Millipore). The reduction (TCEP, Tris (2-carboxyethyl) phosphine) and alkylation (IAA, iodoacetamide) of proteins were performed on 30K 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. The peptide content was quantified by BCA peptide assay (Thermo Fisher) and 25 µg of peptides labeled with Tandem Mass tag (TMT, Thermo Fisher) according to the manufacturer’s recommendation. Labeled peptides were three fractionized by SDB-RPS (poly(styrenedivinylbenzene) reverse phase sulfonate). Detailed protocol and buffer compositions were described at Mann et al. [22] and Lee et al.[23].

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed with Q Exactive (Thermo Fisher) coupled with EASY-nLC1200 UHPLC (Thermo Fisher) as previously reported in our lab [20]. 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.

Isolation and characterization of cancer cell-derived exosomes.

To investigate differences between primary tumor and metastases, we wanted to use tumor cell lines originated from same individual. But no such human breast cancer cell line pairs are available. Thus we used canine mammary gland tumor cell lines; CHMp for primary tumor and CHMm for metastases (derived from metastatic lung pleural effusion) originated from same individual [24]. Canine breast cancer cell lines CHMp and CHMm are used due to its natural occurrence and the gene regulatory sequence of dogs is more similar to humans than mice [19, 25].

We previously established an exosome isolation method to purify exosomes from CHMp and CHMm [19]. The strategy we used is detailed in Fig. 1A. To further characterize the morphology of CHMp- and CHMm-derived exosomes, negative stained exosomes were performed with transmission electron microscope (TEM). The isolated exosomes were composed of 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). The measurement of exosome diameter was performed by dynamic light scattering (DLS). DLS showed the average diameter of exosomes derived from CHMp was within 100 nm, while exosomes derived from CHMm were approximately 33 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 filter aided sample preparation (FASP). Technical replicates consisting of three samples each 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). The correlation among replicates for the CHMp and CHMm exosomal proteins was examined by principal component (PC) analysis which approved a high correlation (> 0.9) (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). Among total 1,284 proteins identified, 87 proteins were enriched in CHMp exosomes by log2 (FC (fold change)) > 1.2 and p-value < 0.05 and 63 proteins in CHMm exosomes by log2(FC) < -1.2 and p-value < 0.05 (Fig. 2F, Supplementary Table 1–3). Those 87 CHMp exosomal proteins were predominantly localized in the extracellular region or nucleus, while 63 CHMm exosomal proteins showed different localization patterns, mainly in the extracellular region or plasma membrane (Fig. 2G). These results suggest significant differences between the component of exosomal proteins derived from primary tumors and metastases, indicating that heterogeneity between primary tumors and metastases was reflected in the composition of exosomal proteins.

Protein interactions identified in CHMp and CHMm exosomal proteins.

To gain a better understanding of the function of the exosomal proteins identified, Gene ontology (GO), STRING (Search Tool for the Retrieval of Interacting Genes) and Gene Set Enrichment Analysis (GSEA) were conducted (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 mostly involved in 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 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). There results indicated 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.

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 [26]. We selected SW480 enriched exosomal proteins with fold change (FC) > 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 commonly 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). 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 LDHA proteins were significantly enriched in primary tumor-derived exosomes cross species and cancers. GPI proteins were enriched only in CHMp-derived exosomes (Fig. 4D). 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 effect on the lactate production of neighboring tumor microenvironment or distant target cells and have implications for cancer prognosis.

Metastases exhibit unique characteristics and high heterogeneity due to the differential TME in which they reside [27, 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.

We conducted a comparative analysis using the exosome proteomic 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 is a key metabolic pathway involved in energy production and biosynthesis in cancer cells [24]. Additionally, glycolysis is closely associated with tumor metastasis [25]. Exosome-mediated metabolic reprogramming has been shown to play a crucial role in tumor microenvironment formation and tumor progression [26]. The reprogramming affects 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. Within the 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 knockout (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, in breast cancer and colorectal cancer has been associated with patient’s poor prognosis. These findings suggest that primary tumor-derived exosomes may influence lactate production in the tumor microenvironment or distant cells, thereby influencing 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 rather 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. 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.

LC-MS/MS Liquid chromatography - mass spectrometry

GPI Glucose phosphate isomerase

LDHA Lactate dehydrogenase A

LDHB Lactate dehydrogenase B

TPI1 Triosephosphate Isomerase 1

ALDOA Aldolase A

TME Tumor microenvironment

FASP Filter-aided sample preparation

SDB-RPS poly(styrenedivinylbenzene) reverse phase sulfonate

TEM Transmission electron microscope

DLS Dynamic light scattering

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

HSP90B1 Heat Shock Protein 90 Beta Family Member 1

HIST2H3A Histone cluster 2 H3 family

SUB1 SUB1 Regulator of Transcription

MAN2A1 Mannosidase Alpha Class 2A Member 1

BTGALT1 β-1, 4-Galactosyltransferase gene

LAMP1 Lysosomal Associated Membrane Protein 1

ITGB5 Integrin Subunit Beta

PPI Protein-protein interaction

STRING Search Tool for the Retrieval of Interacting Genes

GSEA Gene Set Enrichment Analysis

Ethics approval and consent to participate.

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

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

Competing interests

The authors declare no competing interests.

Funding

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) .

Authors’ contributions

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

Acknowledgements

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

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No competing interests reported.

Table1.Totalproteinlist.xlsx
Table2.CHMpexosomalproteinlist.xlsx
Table3.CHMmexosomalproteinlist.xlsx
Profilingandcomparisonofproteomesbetweenprimarytumorandmetastasesderivedexosomes.pdf
floatimage5.png
Supplementary material 1. Full-length Western blot images. (A) Full blot image for figure 4d (up). (B) Full blot image for figure 4d (below).

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You are reading this latest preprint version

Exosome proteomes reveal glycolysis-related enzyme enrichment in primary tumors

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Methods

Cell culture

Exosome isolation

Electron microscopy (EM)

Western blot assay

Proteomics sampling and LC-MS/MS

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1