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Molecular profiling of tumor-derived exosomes in plasma of patients with melanoma

ABSTRACT: By isolation of exosomes from body fluids such as serum or plasma with the use of a gel filtration method developed for this purpose, we showed that the level of exosomes in patients with melanoma is higher and may be associated with progression of the tumor and treatment response. By applying the techniques of mass spectrometry we would like to assess the protein profile of exosomes released by melanoma cells and determine whether these changes correlate with selected clinical parameters. The model tests are exosomes released in vivo from melanoma cells (MMTEX). Proteins of exosomes were subjected to qualitative and quantitative analysis by LC-MS/MS. Proteins were digested by trypsin and were subsequently purified on C18 StageTips which significantly improves the chromatographic separation and allows identification of a higher number of proteins. In addition, after digestion the obtained peptides were fractionated and separated using ion chromatography and reversed-phase chromatography (C18 column), and then analysed by tandem mass spectrometry analysis. To evaluate protein content and verify the presence of exosomes we were used the Western blot technique or flow cytometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma, Extracellular Vesicle

DISEASE(S): Melanoma

SUBMITTER: Monika Pietrowska

LAB HEAD: Piotr Widłak

PROVIDER: PXD021285 | Pride | 2021-08-12

REPOSITORIES: pride

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Project description:The high incidence of pre-eclampsia, affecting 5-10% of pregnancies, makes it a major health problem. Early detection of pre-eclampsia, before the appearance of the first clinical symptoms, is therefore an urgent need, since it would allow the early intervention. Identification of plasma/serum biomarkers would pave the way to develop new strategies for the diagnosis of pre-eclampsia. Liquid biopsy emerges as a promising source of protein biomarkers that circumvent some of the inherent challenges of proteome wide analysis of plasma and serum. Besides, purified exosomes have the added interest of being communication vehicles between cells and tissues both in physiological and pathological processes. Methods We compared the protein abundance in purified exosomes from three different cohorts of control and preeclamptic serum samples, obtained around the sixth month and at the end of pregnancy. To this end, a shotgun label-free proteomics analysis was conducted and the relevant differential proteins were then validated by targeted MRM. Results An exosome purification method was developed that yielded highly enriched preparations, as indicated by the detection of a large number of exosomal markers. Likewise, the presence of specific pregnancy protein markers suggested that a very significant percentage of purified exosomes come from tissues related to pregnancy. Shotgun quantitative proteomic analysis allowed us to identify 10, 114 and 98 differentially regulated proteins in the three studied cohorts, with a high degree of concordance among them. Functional analysis suggests that these proteins participate in biological processes closely related to pre-eclampsia, such as angiogenesis, inflammation or cell migration. Differential abundance of 66 proteins were validated by MRM. Finally, we show the impact of the pre-eclampsia associated exosomes in the proteome of a cellular model, compared to normal exosomes. Conclusions We identified and validated differential proteins in liquid biopsy of patients that open new possibilities for the early diagnostic of pre-eclampsia. Additionally, the functional impact of the different proteome composition of purified pre-eclamptic exosomes in target cells provide new information to further understand changes on embryo-maternal interactions and therefore the pathogenesis of this disease.

Project description:Intra-tumor heterogeneity (ITH) results from coexistence of genetically distinct cancer cell (sub)populations, their phenotypic plasticity, and the presence of heterotypic components of the tumor microenvironment (TME). Here we addressed potential association between phenotypic ITH revealed by mass spectrometry imaging (MSI) and the prognosis of breast cancer. Tissue specimens resected from 59 patients treated radically due to the locally advanced HER2-positive invasive ductal carcinoma were included in the study. After on-tissue trypsin digestion of cellu-lar proteins, peptide maps of all cancer regions (about 380,000 spectra in total) were segmented by an unsupervised approach to reveal their intrinsic heterogeneity. A high degree of similarity between spectra was observed, which indicated relative homogeneity of cancer regions. How-ever, when the number and diversity of the detected clusters of spectra were analyzed, differ-ences between patient groups were observed. It is noteworthy that a higher degree of heteroge-neity was found in tumors from patients who remained disease-free during a 5-year follow-up (n=38) compared to tumors from patients with progressive disease (distant metastases detected during follow-up, n=21). Interestingly, such differences were not observed between patients with different status of regional lymph nodes, cancer grade, or expression of estrogen receptor at the time of the primary treatment. Subsequently, spectral components with different abundance in cancer regions were detected in patients with different outcomes, and their hypothetical identity was established by assignment to measured masses of tryptic peptides identified in correspond-ing tissue lysates. Such differentiating components were associated with proteins involved in immune regulation and hemostasis. We postulate that a higher heterogeneity of tumors with a better prognosis could reflect the presence of heterotypic components including the microvas-culature and infiltrating immune cells, that facilitated response to treatment.

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Molecular profiling of tumor-derived exosomes in plasma of patients with melanoma

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