Agnieszka Szyposzynska

Comparative Analysis of Primary Ovarian Cancer Cells and Established Cell Lines as a New Tool for Studies on Ovarian Cancer Cell Complexity

Primary cancer cells reflect the genetic background and phenotype of a tumor. Immortalized cells with higher proliferation activity have an advantage over primary cells. The aim of the study was to immortalize the primary ovarian cancer (OvCa) cells using the plasmid-carrying human telomerase reverse transcriptase (hTERT) gene and compare their phenotype and biological activity with the primary cells. The primary OvCa3 A and OvCa7 A cells were isolated from the ascitic fluid of two high-grade serous ovarian cancer patients and were characterized using immunocytochemical methods, flow cytometry, real-time RT-PCR, Western blot, metabolic activity, and migratory potential. Both immortalized ovarian cancer cell lines mirrored the phenotype of primary cancer cells, albeit with modifications. The OvCa3 A hTERT cells kept the mesenchymal stem cell phenotype of CD73/CD90/CD105-positivity and were CD133-negative, whereas the cell population of OvCa7 A hTERT lost CD73 expression, but almost 90% of cells expressed the CD133 characteristic for the CSCs phenotype. Immortalized OvCa cells differed in gene expression level with respect to Sox2 and Oct4, which was associated with stemness properties. The OvCa7 A hTERT cells showed higher metabolic and migratory activity and ALDH1 expression than the corresponding primary OvCa cells. Both primary and immortalized cell lines were able to form spheroids. The newly established unique immortalized cell line OvCa7 A hTERT, with the characteristic of a serous ovarian cancer malignancy feature, and with the accumulation of the p53, Pax8, and overexpression of the CD133 and CD44 molecules, may be a useful tool for research on therapeutic approaches, especially those targeting CSCs in ovarian cancer and in preclinical 2D and 3D models.

Mesenchymal Stem Cell Microvesicles from Adipose Tissue: Unraveling Their Impact on Primary Ovarian Cancer Cells and Their Therapeutic Opportunities

Mesenchymal stem cells (MSCs) and their derivatives can be promising tools in oncology including ovarian cancer treatment. This study aimed to determine the effect of HATMSC2-MVs (microvesicles derived from human immortalized mesenchymal stem cells of adipose tissue origin) on the fate and behavior of primary ovarian cancer cells. Human primary ovarian cancer (OvCa) cells were isolated from two sources: post-operative tissue of ovarian cancer and ascitic fluid. The phenotype of cells was characterized using flow cytometry, real-time RT-PCR, and immunofluorescence staining. The effect of HATMSC2-MVs on the biological activity of primary cells was analyzed in 2D (proliferation, migration, and cell survival) and 3D (cell survival) models. We demonstrated that HATMSC2-MVs internalized into primary ovarian cancer cells decrease the metabolic activity and induce the cancer cell death and are leading to decreased migratory activity of tumor cells. The results suggests that the anti-cancer effect of HATMSC2-MVs, with high probability, is contributed by the delivery of molecules that induce cell cycle arrest and apoptosis (p21, tumor suppressor p53, executor caspase 3) and proapoptotic regulators (bad, BIM, Fas, FasL, p27, TRAIL-R1, TRAIL-R2), and their presence has been confirmed by apoptotic protein antibody array. In this study, we demonstrate the ability to inhibit primary OvCa cells growth and apoptosis induction after exposure of OvCa cells on HATMSC2-MVs treatment; however, further studies are needed to clarify their anticancer activities.

Suppression of Ovarian Cancer Cell Growth by AT-MSC Microvesicles

Transport of bioactive cargo of microvesicles (MVs) into target cells can affect their fate and behavior and change their microenvironment. We assessed the effect of MVs derived from human immortalized mesenchymal stem cells of adipose tissue-origin (HATMSC2-MVs) on the biological activity of the ovarian cancer cell lines ES-2 (clear cell carcinoma) and OAW-42 (cystadenocarcinoma). The HATMSC2-MVs were characterized using dynamic light scattering (DLS), transmission electron microscopy, and flow cytometry. The anti-tumor properties of HATMSC2-MVs were assessed using MTT for metabolic activity and flow cytometry for cell survival, cell cycle progression, and phenotype. The secretion profile of ovarian cancer cells was evaluated with a protein antibody array. Both cell lines internalized HATMSC2-MVs, which was associated with a decreased metabolic activity of cancer cells. HATMSC2-MVs exerted a pro-apoptotic and/or necrotic effect on ES-2 and OAW-42 cells and increased the expression of anti-tumor factors in both cell lines compared to control. In conclusion, we confirmed an effective transfer of HATMSC2-MVs into ovarian cancer cells that resulted in the inhibition of cell proliferation via different pathways, apoptosis and/or necrosis, which, with high likelihood, is related to the presence of different anti-tumor factors secreted by the ES-2 and OAW-42 cells.