Flow Cytometry Analysis of Surface PD-L1 Expression Induced by IFNγ and Romidepsin in Ovarian Cancer Cells

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.

Download Full-text

LY294002 and Metformin Cooperatively Enhance the Inhibition of Growth and the Induction of Apoptosis of Ovarian Cancer Cells

International Journal of Gynecological Cancer ◽

10.1097/igc.0b013e3182322834 ◽

2012 ◽

Vol 22 (1) ◽

pp. 15-22 ◽

Cited By ~ 28

Author(s):

Cuilan Li ◽

Vincent Wing Sun Liu ◽

David Wai Chan ◽

Kwok Ming Yao ◽

Hextan Yuen Sheung Ngan

Keyword(s):

Cell Cycle ◽

Ovarian Cancer ◽

Flow Cytometry ◽

Cell Growth ◽

Cancer Cells ◽

Cancer Cell ◽

Ovarian Cancer Cell ◽

Mtor Pathway ◽

Ovarian Cancer Cells ◽

Cancer Cell Growth

BackgroundThe phosphoinositide 3 kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)/mammalian target of rapamycin (mTOR) pathway is frequently aberrantly activated in ovarian cancer and confers the chemoresistant phenotype of ovarian cancer cells. LY294002 (PI3K inhibitor) and metformin (5′-adenosine monophosphate [AMP]-activated protein kinase [AMPK] activator) are 2 drugs that were known to inhibit mTOR expression through the AKT-dependent and AKT-independent pathways, respectively. In this study, we explored the effectiveness of LY294002 and metformin in combination on inhibition of ovarian cancer cell growth.MethodsWestern blotting was used to detect the changes of PI3K/AKT/mTOR and AMPK/acetyl-CoA carboxylase (ACC) signaling activities, cell cycle control, and apoptosis. Cell growth was evaluated by cell proliferation, colony formation, and soft agar assays. Flow cytometry was used to study cell cycle distribution and cell death upon drug treatment.ResultsOur study showed that LY294002 and metformin in combination could simultaneously enhance the repression of the PI3K/AKT/mTOR pathway and the activation of the AMPK/ACC pathway. The downstream target of AKT and AMPK, mTOR, was cooperatively repressed when the drugs were used together. The cell cycle regulatory factors, p53, p27, and p21, were up-regulated. On the other hand, caspase 3 and poly (ADP-ribose) polymerase activities involved in apoptosis were also activated. Cell growth assays indicated that LY294002 and metformin could effectively inhibit ovarian cancer cell growth. Flow cytometry analysis showed that the treatment of the 2 drugs mentioned above induced cell cycle arrest at G1 phase and increased sub-G1 apoptotic cells.ConclusionThe combinational use of LY294002 and metformin can enhance inhibition of the growth and induction of the apoptosis of ovarian cancer cells. Our results may provide significant insight into the future therapeutic regimens in ovarian cancer.

Download Full-text

Niraparib exhibits a synergistic anti-tumor effect with PD-L1 blockade by inducing an immune response in ovarian cancer

Journal of Translational Medicine ◽

10.1186/s12967-021-03073-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Jinyu Meng ◽

Jin Peng ◽

Jie Feng ◽

Jochen Maurer ◽

Xiao Li ◽

...

Keyword(s):

Ovarian Cancer ◽

Flow Cytometry ◽

Immune Response ◽

T Cells ◽

Cancer Cells ◽

Parp Inhibitors ◽

Ovarian Cancer Cells ◽

Human Ovarian Cancer ◽

Sting Pathway

Abstract Background Immune checkpoint blockades (ICBs) therapy showed limited efficacy in ovarian cancer management. Increasing evidence indicated that conventional and targeted therapies could affect tumor-associated immune responses and increase the effectiveness of immunotherapy. However, the effects of Niraparib, one of the poly (ADP) ribose polymerase (PARP) inhibitors, on the immune response remains unclear. Delineating the crosstalk between cytotoxic anticancer agents and cancer-associated immunity may lead to more efficient combinatorial strategies. Methods Programmed death ligand 1 (PD-L1) expression in human ovarian cancer cells after PARP inhibitors treatment was examined by western blotting (WB) and flow cytometry. The expression of poly ADP-ribose polymerase (PARP1), PD-L1, and CD8 in human ovarian cancer tissues was detected by immunohistochemistry(IHC). The effect of Niraparib and PD-L1 blockade in ovarian cancer progression was investigated in vivo. The changes of immune cells and cytokines in vitro and in vivo were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Changes of cGAS/STING signal pathway after Niraparib treatment were determined by WB, ELISA. Results Niraparib upregulated membrane PD-L1 and total PD-L1 expression in ovarian cancer cells and had a synergistic effect with PD-L1 blockade in vivo. In clinical patient samples, Niraparib augmented cytotoxic CD8+T cell proportion and function. In vivo and vitro, Niraparib can also increase the proportion of T cells and combined with PD-L1 blockade could further enhance the effect. Besides, Niraparib activated the cGAS-STING pathway, increasing the levels of cytokines such as CCL5 and CXCL10, which played a vital role in augmenting the infiltration and activation of cytotoxic T cells. Conclusions Niraparib could modulate the immune response via the activation of the cGAS/STING pathway, and combination with PD-L1 blockade could further enhance the effect. These results provide a sound theoretical basis for clinical treatment.

Download Full-text

Interferons alpha and gamma with monocytes as a therapeutic strategy for ovarian cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.5_suppl.9 ◽

2020 ◽

Vol 38 (5_suppl) ◽

pp. 9-9

Author(s):

Christina M. Annunziata ◽

Anna Duemler ◽

Franklin Ning

Keyword(s):

Ovarian Cancer ◽

Flow Cytometry ◽

Cancer Cells ◽

Tumor Cells ◽

Interferon Alpha ◽

Immune Cell ◽

Ovarian Cancer Cells ◽

Human Monocytes ◽

Differentiation Markers ◽

Monocyte Differentiation

9 Background: In the presence of pro-inflammatory cytokines, monocytes are cytotoxic to tumor cells. We previously showed that monocytes stimulated with interferon alpha and gamma result in synergistic killing of ovarian cancer cells in vitro. Here we better characterize monocyte differentiation and their ability to induce cell death through co-culture experiments with spheroids and in mouse xenografts. Methods: OVCAR8 cells were grown in ultra-low attachment conditions for three days before being co-cultured with human monocytes as well as interferon gamma and interferon alpha. Monocytes and OVCAR8s were assayed by flow cytometry for markers of differentiation and viability, respectively. Mouse studies were performed to confirm the viability of human monocytes stimulated with interferon alpha and gamma within the peritoneal cavity seventy-two hours after injection. Subsequent mouse experiments analyzed monocyte differentiation towards M1 or M2 phenotypes by flow cytometry with or without exposure to tumor cells and/or interferons. Results: OVCAR8 spheroids showed decreased viability in the presence of monocytes combined with interferon. We show that monocytes also express a hybrid M1/M2 phenotype when stimulated to IFN and exposed to tumor cells. In mice, monocytes demonstrated a unique differentiation towards both M1 and M2 macrophages as well as plasmacytoid and classic DCs. Various immune cell subsets also differed when comparing peritoneal washes versus tissue digests. Mouse survival studies are ongoing. Conclusions: Monocytes with the combination of interferons alpha and gamma are effective at killing ovarian cancer cells in laboratory models. Phenotypic analyses show a novel pattern of differentiation markers. Our ongoing clinical trial with these agents will incorporate similar studies to correlate monocyte differentiation with anti-tumor activity.

Download Full-text