Tesis

Dampak pemberian vesikel ekstraseluler sel punca mesenkim jaringan tali pusat terhadap kepuncaan sel kanker payudara (MDAMB-231, MCF7, dan ALDH+) = The effect of mesenchymal stem cells derived extracellular vesicles supplementation on the stemness of breast cancer cells (MDA-MB- 231, MCF7, and ALDH+).

Sel punca mesenkim (SPM) banyak dikembangkan untuk terapi penyakit degeneratif. Namun, SPM yang dapat direkrut ke lingkungan mikrotumor dapat bersifat pro- maupun antitumorigenik, melalui vesikel ekstraseluler (VE) yang disekresikannya. Sel punca kanker (SPK) merupakan populasi kecil sel penyusun jaringan kanker yang dikaitkan dengan resistensi terapi dan metastasis. Penelitian terdahulu menyatakan bahwa CM SPM tali pusat meningkatkan kepuncaan sel kanker payudara. Saat ini belum diketahui peran VE SPM tali pusat terhadap kepuncaan sel kanker payudara. Penelitian ini bertujuan menganalisis dampak pemberian VE SPM tali pusat terhadap kepuncaan sel kanker payudara. VE diisolasi dari CM SPM tali pusat dengan teknik kromatografi kolom. Identifikasi VE dilakukan dengan mikroskop konfokal dan transmission electron microscope (TEM). Internalisasi VE oleh sel kanker payudara dikonfirmasi dengan pengamatan mikroskop konfokal. Analisis viabilitas sel pasca kokultur VE dilakukan menggunakan trypan blue exclusion assay. Analisis ekspresi mRNA OCT4 dilakukan dengan qRT-PCR (metode Livak). Ekspresi protein OCT4 dianalisis dengan metode Western Blot. Aktivitas enzim ALDH dianalisis dengan ALDEFLUOR . Hasil yang diperoleh adalah VE SPM tali pusat berhasil diisolasi dengan metode kromatografi kolom serta diidentifikasi dengan mikroskop konfokal dan TEM. Sel MDA-MB-231, MCF7, dan ALDH+ menginternalisasi VE SPM tali pusat dengan derajat yang berbeda. VE SPM tali pusat 5% meningkatkan viabilitas sel MDA-MB-231, MCF7, dan ALDH+. Tren peningkatan ekspresi mRNA OCT4 ditemukan pada semua sel kelompok VE 5%. Tingkat ekspresi protein OCT4 sel MCF7 dan ALDH+ berbanding terbalik dengan peningkatan konsentrasi VE. Ekspresi protein OCT4 sel MDA-MB-231 meningkat pasca suplementasi VE 5%. VE 5% meningkatkan aktivitas ALDH ketiga sel kanker payudara. VE 10% menurunkan aktivitas ALDH sel MDA-MB- 231 dan MCF7 namun meningkatkan aktivitas ALDH sel ALDH+. Sebagai kesimpulan, pemberian VE SPM tali pusat dengan konsentrasi yang berbeda dapat memberikan dampak yang berbeda terhadap kepuncaan berbagai sel kanker payudara, berkaitan dengan regulasi ekspresi OCT4 dan aktivitas ALDH.
Kata kunci: Vesikel ekstraseluler, SPM, tali pusat, kepuncaan, kanker payudara


Mesenchymal Stem Cells (MSCs) are currently developed for regenerative medicine. However, MSCs can also be recruited into the tumor microenvironment to modulate cancer progression by releasing extracellular vesicles (EVs). Cancer stem cell is a minor subpopulation of cells within heterogenous cancerous tissue that had been associated with therapy resistance and metastasis.. The aim of this study was to investigate the effect of umbilical cord MSC (UCMSC-EVs) on the viability and expression of stemness markers of human MDA-MB-231, MCF7, and ALDH+ breast cancer cells. UCMSC-EVs were successfully isolated from the conditioned medium ofUCMSCs using size exclusion chromatography (SEC). The isolated EV fractions were stained with PKH26 fluorescence for confocal microscopy analysis. Moreover, the presence of EVs was verified under a transmission electron microscope. Five and ten percent (v/v) concentration of UCMSC-EVs were then co-cultured with human MDA-MB-231, MCF7, and ALDH+ cells. To investigate UCMSC-EV uptake by MDA-MB-231, MCF7, and ALDH+ cells, confocal microscopy analysis was performed. Subsequently, the viability of co-cultured breast cancer cells was determined using trypan blue exclusion assay, while their mRNA and protein expression of OCT4 as well as ALDH activity as the marker of stemness properties were analyzed using quantitative reverse transcription polymerase chain reaction, Western Blot, and ALDEFLUOR , respectively. As the result, UCMSC-EVs were successfully isolated using SEC technique. The internalization ability of each type of breast cancer cells seemed different. Notably, UCMSC-EVs co-culture at 5% concentration significantly increased the viability of MCF7 and MDA-MB-231 cells. The increasing of OCT4 mRNA expression could be detected in the group of cells supplemented with 5% EVs. On MCF7 and ALDH+ cells, we found that the higher the EVs concentration given, the lower expression of OCT4 protein was. The highest OCT4 protein expression of MDA-MB-231 cells was detected in the supplementation of 5% EVs. Supplementation of 5% EVs increased the ALDH activity within three types of cells. However, at 10% concentration, MSC-EVs reduced the ALDH activity of MCF7 and MDA-MB-231 cells. In conclusion, supplementation of UCMSC-EVs in different concentrations gives different impacts in terms of stemness that is correlated with OCT4 and ALDH regulation within the treated cells.
Keywords: Vesicle extracellular, mesenchymal stem cells, umbilical cord, stemness, breast cancer.