Effects of exosomes isolated from PID-PCs on pankreatic cancer

The pancreas consists of two parts, endocrine and exocrine. While the exocrine pancreas includes acinar and canal cells, the endocrine pancreas consists of the Langerhans islet which has five different types of cells. Pancreas cancer is one of the deadliest malignant neoplasms and the leading cause of death because of cancer. The annual survival rate is less than 5% for pancreatic cancer, which is also late in its diagnosis due to the fact that the symptoms occur at the advanced stage and are not distinguishing. A stem cell has the capability of sell perpetuation, clonality, and differentiation and it plays a role constitution of tissue and organ in the embryonic and fetal period, it is also effective in regeneration in the adult period. Mesenchymal stem cell which is one of the adult period stem cells has the capability of antiinflammatory, immunoregulatory, and immunosuppressive. Pancreatic islet-derived progenitor cells (PID-PC) has also properties of typically mesenchymal stem cells. The size of exosomes is 50- 150 nm, and are vesicles encased in a lipid bilayer. These vesicles that its effective cell-to-cell communication differs from other vesicles due to their RNA cargo contents. The fact that they can cause changes in the target cell by carrying miRNA and mRNA from the cells they are secreted suggests that exosomes may play a role in the diagnosis and treatment of many different diseases, especially cancer. The purpose of our study is to characterize exosomes isolated from the PID-PC, MiaPaca-2, and INS-1 cell lines and make an analysis comparatively of the size of this exosome for the first time. Besides, the second aim of this study is to research the effects of exosomes which were first time isolated from PID-PC on the INS-1 cells, which is a beta cell tumor, and MiaPaca-2 cells which is a pancreatic cancer EFFECTS OF EXOSOMES ISOLATED FROM PID-PCs ON PANCREATİC CANCER xx cell line. Exosomes were isolated from all three cell lines with exosome isolation kits. Alix, TSG101, CD9, and CD81 proteins which are known as markers for exosomes were analyzed with Simple Wes in cell lysates which were obtained from the three cell lines and also exosomes isolated from cell lines. The size and concentration of exosomes that were isolated from three different cell lines were detected with Nanoparticle Tracking Analysis (NTA). The cell viability test (MTT) was done and IC50 values were detected after exosomes were isolated from PID PC and characterized and also applied to INS-1 and MiaPaca-2 cell lines in which concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 7.5 and 10 µg/ml for 24 and 72 hour. The cells were fixed and were marked immunohistochemically with HSP90, HSF-1, Nestin, Endoglin, Kaspaz-8, Active Kaspaz-3, Beclin, and p-Bcl-2 antibodies after exosomes were applied with determined IC50 value in INS-1 and MiaPaca-2 cell lines for 72 hours. The labeling intensities of the proteins were calculated using the computer-aided image analysis program NIS-Element D 3.1 under the light microscope. The cell cytotoxicity was measured by using an LDH kit after exosomes were applied with a determined IC50 value in the MiaPaca-2 cell line for 72 hours. Moreover, to investigate the functional effect of exosomes on the INS-1 cell line, cell secretion was collected, and cell lysates were obtained after exosomes were applied with a determined IC50 value in INS-1 for 72 hours. Insulin levels were analysed at by using an Insulin ELİSA kit in the obtained cell lysates and secretion. As a result of Simple Wes analysis, Alix protein was detected in exosomes isolated from PID PC, MiaPaca-2, INS-1, and cell lysates obtained from MiaPaca-2, INS-1. TSG101 proteins were detected in cell lysates obtained from PID-PC, MiaPaca-2, and INS-1, but not isolated exosomes. CD9 protein was shown in PID-PC, MiaPaca-2, and INS-1 cell lysates and exosomes isolated from INS-1. On the other hand, CD81 protein was detected only in lysate obtained from the MiaPaca-2 cell line. As a result of NTA, the dimensions of exosomes isolated from PID-PCs as 103.6 ± 28.6 nm, exosomes isolated from MiaPaca-2 cell line as 100.7 ± 10 nm, and exosomes isolated from INS-1 cell line as 147.2 ± 12.3 nm were analyzed. According to cell viability analysis, incubation of MiaPaca-2 cells with exosomes isolated from PID-PCs for 24 hours was not statistically significant compared to the control group at low concentrations of exosome application. As a result of the incubation of MiaPaca-2 cells with isolated exosomes for 72 hours, a decrease in cell viability occurred beginning from low doses. In the INS-1 cell line, there was no difference in cell viability compared to the control until the application of the exosome at a concentration of 5 µg/ml after 24 hours of incubation with the exosome. As a result of incubation of INS-1 cells with exosomes for 72 hours, no change was observed until the application of exosomes at a concentration of 2.5 µg/ml, while cell viability began to decrease as a result of exosome application at higher doses. According to the results of immunohistochemical staining, the immunostaining intensity of HSP90 when exosome was applied to the INS-1 cell line was not statistically significant compared to the control group, while the immunostaining intensity of HSP90 increased significantly in MiaPaca-2 cell line, which was applied to the exosome, compared to the control group. In addition, the HSP90 immunostaining intensity of the MiaPaca-2 cell line was significantly higher than the INS-1 cell line in the exosome-treated groups of both cell lines. While there was no statistically significant difference in HSF-1 immunostaining intensity in the exosome-treated INS-1 and MiaPaca-2 cell lines compared to the control group, the HSF-1 immunostaining intensities were significantly higher in the control group of MiaPaca-2 cell line xxi compared to the control group of the INS-1 cell line, and also the exosome-treated MiaPaca-2 cell line compared to the exosome-treated INS-1 cell line. There was no significant difference in Nestin and Endoglin immunostaining intensity in INS-1 and MiaPaca-2 cell lines treated with and without exosomes. As a result of Nestin immunostaining, there is no labeling of Nestin protein in the control group of both cell lines. Similarly, as a result of Endoglin immunostaining, there was no labeling of Endoglin protein in the INS-1 cell line both in the control group and in the exosome treated group, and in the MiaPaca-2 cell line only in the control group. In the Miapaca-2 cell line, Caspase-8 and Active Caspase-3 immunostaining intensities were not different in the exosome-treated group compared to the control group. There was a statistically significant decrease in Beclin and p-Bcl-2 immunostaining intensities in the exosome-treated MiaPaca-2 cell line compared to the control group. After exosome application to the MiaPaca-2 cell line, the level of LDH released from the MiaPaca-2 cell line was analyzed as 13.98 ± 3.361% and cell cytotoxicity was determined. According to the insulin levels measured with the ELISA kit in the cell secretion and lysate of the exosome-treated INS-1 cells, the insulin level in the cell lysates increased statistically significantly compared to the control group. However, the level of insulin in cell secretion did not differ compared to the control group. In addition, insulin levels in exosome-treated INS-1 cell lysates showed a statistically significant increase compared to cell secretion. As a result, we saw that the exosomes isolated from PID-PC, MiPaca-2, and INS-1 cell lines for the first time by us and whose size analyses were performed, were similar according to the exosome sizes isolated from different sources in the literature. We observed that the Alix protein may be a specific marker for exosomes isolated from the three cell lines and the CD9 protein may be a specific marker only for INS-1 isolated exosomes. In cell viability analysis, we detected that exosome isolated from the PID-PC cell line caused cell death in the MiaPaca 2 cells in a time- and dose-dependent manner. In addition, the IC50 value determined for MiaPaca-2 cells has no effect on cell viability in INS-1 cells, which best mimics pancreatic beta cells and can be used instead of healthy pancreatic beta cells in some studies, suggesting that isolated exosomes can kill cancer cells without damaging healthy cells. The significant increase of HSP90 in the MiaPaca-2 cell line as a result of immunostaining suggests that the increase of this protein may be a marker in the case of possible exosome treatment in pancreatic cancer. A significant decrease in the p-Bcl-2 level in the MiaPaca-2 cell line indicates that cells can die in an apoptotic way, while a significant change in the Active Caspase-3 level indicates that apoptotic cell death is not too much. While the significant decrease in Beclin-1 indicates that the cells may not die autophagically, the LDH level measured in MiaPaca-2 cells indicates that some of the cells that die also die by necrosis. A significant increase in insulin level after exosome administration in INS-1 cells indicates that exosomes increase their production by affecting the insulin mechanism, while the absence of a change in insulin secretion indicates that exosomes do not affect insulin secretion or that there may be problems during the release of excess insulin.