| Background and Objectives:Tumourigenesis is a gradual process involving multi-stage reactions and an accumulation of mutations. Metastasis is an important characteristic in distinguishing between malignant and benign tumours and is closely related to thetumour microenvironment. The tumour microenvironment is composed of tumour cells, stromal cells, tumour-associated immune cells, cytokines and chemokines, and the surrounding microenvironment can be modified by tumour cells, allowing tumour cells to control self-renewal and differentiation processesduringtumour development. Tumour cells promote tumour growth by maintaining and changing their environmentvia autocrineand paracrinesignalling. In the tumour microenvironment, some of the main molecules that mediate communicationbetween cells are secreted proteins, which are complex products secreted by a variety of cells. Communication between cells occurs when these secreted proteins bind to cell surface receptors; however, the cells also secrete more complex structures called membrane vesicles, which consist of transmembrane proteins embedded in a lipid bilayer and wrapped in a hydrophilic component.Exosomes are membranous vesicles 30-100 nm in diameter thatare released through the fusion of multivesicular bodies with the plasma membrane and are classified as a type of secretory vesicle. Exosomes secreted by various cells can contain miRNA, mRNA and cytoplasmic proteins, which are responsible for mediating communication between cells. Tumour-derived exosomes are present inculture supernatants andin the plasma of patients with tumours or in malignant exudates and affect the progression of tumours by influencing the tumour microenvironment, promoting tumour angiogenesis and tumour metastasis, or directly acting on the tumour cells, among other mechanisms. In addition, identifying specific exosomes consisting of tumour-associated proteins and RNA could result in these vesicles becoming a new source of biomarkers. Recently, exosomes have been isolated and studied from manycell lines and body fluids. These findings show that exosomes are present in most body fluids and may thus be a valuable tool for monitoring a patient’shealth state.The scarcity of exosome studies can be attributed to the traditional exosome extraction process being relatively complex and cumbersome. Therefore, our first aim was to establish and validate aset of methods for simple and efficient extraction of tumour-derived exosomes.TP53 is one of the most studied genes, owing to its high correlation with human tumours. Initially, TP53 was considered to be a oncogene, but upon further studyTP53 has been widely recognised as a tumour suppressor gene. The p53 protein has a very wide range of functions, including cell cycle arrest, induction of apoptosis and senescence, maintenance of genetic stability, regulation of angiogenesis, cell differentiation and immune reactivity. TP53 mutations occur in approximately 50%of tumours. WhenTP53 is mutated, the p53 protein will lose its function as atumour suppressorwhile potentially gainingseveral oncogenic features. Clinical trials have demonstrated that mutations in the TP53 gene are closely related to the occurrence of tumours, and the TP53 gene has thus become a hotsubject for research effortsinto understanding the occurrence and development of tumours. Given that TP53 is frequently mutatedin tumours, that mutant TP53 promotes the development of tumours and that tumour development is inseparable from the tumour microenvironment, it is essential to determine the effect of mutant TP53 on the tumour microenvironment. Althoughhow mutant TP53 affects the biological behaviour of tumour cells is already well understood, the effect ofTP53 mutations on the secretion of proteinsfrom tumour cells into the tumour microenvironment is poorly understood. In addition to classical protein secretion, cells also release secreted proteins via exosomes. Exosomes are one of the main non-classical ways to release secreted proteins.. Therefore, another purpose of this study was to compare differences in exosome protein composition between the TP53 mutant, TP53 knock-out and wild-type tumour cells and to investigate the effect of mutant TP53 on secreted proteins in the tumour microenvironment.Methods:A new nanomaterial was used to isolate exosomes from the culture supernatant of Huh7 hepatoma cells. We observed Huh7 cell morphology by transmission electron microscopy, detectedexosome marker proteins by Western blotting, and compared Huh7 exosomal protein concentration and morphology using both a traditional ultracentrifugation method as well as the nanomaterials method. Furthermore, two potential hepatoma-associated proteins, tissue transglutaminase 2 and annexin A2, were analysed in exosomes from the culture supernatant of Huh7 cells by Western blotting. With these experiments, we provide a proof-of-concept for the use of a new nanomaterial in the detection of non-classical secretory pathway proteins.We constructed a TP53 mutant version of the colon cancer cell lineHCT116 and employed new nanomaterials to extract exosomes from the culture supernatant of this TP53 mutant line, as well as from TP53 wild-type and TP53 knock-out HCT116 cells. Exosome morphology was visualised by transmission electron microscopy, and exosome marker proteins were identified by Western blotting to determine whether the nanomaterials could achieve fast and efficient separation of exosomes. Next, we calculated the diameters of 200 exosomes that were randomly selected from several fields of view under an electron microscope, and compared the sizes of exosomes derived from each of the three cell lines. Finally, exosome protein composition differences were analysedusing iTRAQ-LC-MS/MS,and differences in protein expressionwere verified by ELISA.Results:Part 1:The exosomes observed by transmission electron microscopy showed disk-shaped membrane vesicles with diameters between 30-100 nm. Protein markers with positive staining on the surfaces of exosome membranes included the transmembrane protein CD63 and the heat shock protein HSP70. The concentration of exosomal proteins extracted by the new nanomaterials method was approximately 780 μg/108 cells, which was 19 times higher than the concentration obtained from exosomes extracted in the traditional manner. Exosomes secreted by hepatoma cells also contained the hepatoma-associated protein TGM2 but were negative for annexin A2.Part 2:The new nanomaterial effectively enriched for exosomes, and exosomes from wild-type TP53 cells were significantly larger in size than those from the TP53 mutant and TP53 knock-out cells. We compared exosomal proteins derived from TP53 wild-type HCT116 cells and TP53 mutant HCT116 cells and identified 144 differentially expressed proteins. When comparing between the TP53 wild-type HCT116 cells and the TP53 knockout HCT116 cells,480 differential proteins were found. Most of the differential proteins are involved in metabolism, cellular processes, immune processes, apoptosis, stimulus response, reproduction and cell adhesion. Ingenuity pathway analysis (IPA) software analysis showed that differential proteins were enriched for protein ubiquitination pathways, mTOR signalling and DNA break repair in the analysis comparing TP53 wild-type HCT116 cells and the TP53 mutant HCT116 cells, withparticular enrichment for repair factors and signal transduction molecules. IPAanalysis comparing TP53 wild-type HCT116 cells and the TP53 knockout HCT116 cells showed enrichment in protein ubiquitination pathways, EIF2 signalling and the glycolytic pathway. Furthermore, the serum level of RBI in colon cancer patients was significantly higher than inhealthy patients (P= 0.0012).Conclusion:Part 1:The new extraction method based on nanomaterials is quick and efficient. The hepatoma-associated protein TGM2 can be secreted through an exosome-mediated non-classical secretion pathway, and it may be a valuable tumour marker.Part 2:Both TP53 mutation and knock-out altered the protein composition of exosomes and enriched for many ubiquitinated proteins. TP53 mutations may presumably affect exosome ubiquitinated proteinsto further regulate other contents loaded intosorting exosomes. Additionally, TP53 mutations may stimulate cells to utilize the non-classical exosomal secretory pathway, contributing to tumour cell growth.These results warrant further investigation into the influence of TP53 mutationson thetumour microenvironment. In addition, our finding that serum levels of RB1 in patients with colon cancer were significantly higher than in healthy controlsis consistent with previous reports and with our quantitative iTRAQ results. We speculate that exosomes expressing high levels of RBI act as a signal betweentumour cells,inhibitingtumour cell apoptosisand thereby promoting tumour progression.The main innovation:1. When extracting exosomes, most of the literature adopts traditional methods, such as ultracentrifugation and sucrose density gradient centrifugation, which are time-consuming. In this paper, we apply a novel method that involves a simple and efficient procedure for separation and extraction of exosomes. This method has a short exposure time in an open system, preventing contamination from the environment and from cell debris.2. We extracted exosomes from TP53-mutated, TP53 knock-out and TP53 wild-type tumour cells and conducted an analysis of differentially expressed proteins, employing ELISA for validation of protein expression levels. We conductedthis preliminary study investigating the effect of TP53 state on thetumour microenvironment as a basis for future experiments in other systems. |
