Characterization of the tumor microenvironment of ovarian cancer patients in relation to lymphocytes subtypes, cytokines and functional activation of natural killer cells

Author(s)

Rodrigo Fernandes da Silva

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2018-02-27

Examining board members

Sophie Françoise Mauricette Derchain; Luís Otávio Zanatta Sarian; Luiz Gustavo de Almeida Chuffa; Ana Paula Lepique; Maria Laura Costa do Nascimento

• Advisor: Sophie Françoise Mauricette Derchain

Abstract

Peritoneal ascites is a distinguishable feature of patients with advanced epithelial ovarian cancer (EOC), and is a result of the inflammatory response triggered by the malignancy. The presence of different lymphocyte subsets has been reported to exert activatory and inhibitory function on the immune response against the tumor in this microenvironment. Objectives: Characterize the ascites liquid from ovarian cancer patients in relation to lymphocyte subtypes, cytokines and functional activation of NK cells. Describe the phenotypic and molecular characteristics of the ovarian cancer cell line CAISMOV24 and compare to the genomic alterations between the cell line and their primary malignant cells. Methodology: Ascites were collected by paracentesis guided by ultrasonography and the blood by venipuncture. The samples were processed to obtain the cell fraction and supernatant/serum, which were stored in liquid nitrogen and -20oC respectively. The cell fraction samples were for the phenotyping of lymphocyte subtypes, evaluation of NK cells activating receptors expression and NK cell functional activity, measured by the degranulation marker CD107a. The supernatant/serum samples were used for the quantification of cytokines. All the analyzes were done by flow cytometry. The CAISMOV24 cell line was established by cells isolated from malignant ascites from a patient with ovarian cancer. It was evaluated its cellular expansion potential, production of soluble biomarkers, expression of surface molecules and genetic mutations typical of ovarian serous carcinoma. Additionally, comparative genomic hybridization was used to compare genomic alterations between CAISMOV24 and its primary malignant cells. Results: NK cells degranulation from EOC cell-free ascites was significantly (p<0,05) higher than all the other groups, either in their resting state or after IL-2 stimulation, suggesting a previous local stimulation. In contrast, the stimulation with IL-2 had no effect on NK cells from ascites with EOC cells. The amount of T-regs and the cytokine TNF-? (tumor necrosis factor-?) was significantly higher in ascites with EOC cells compared to EOC cell-free ascites, suggesting immunomodulation and inflammation related to the tumor malignancy. The CAISMOV24 cell line has been in continuous culture for more than 30 months and more than 100 in vitro passages. The cell surface molecules EpCAM, PVR and CD73 are overexpressed on CAISMOV24 cells compared to the primary malignant cells. CAISMOV24 continued to produce CA125 and HE4 in vitro. Although the cell line had developed alongside the accumaltion of genomic alterations (28 CNV in primary malignant cells and 37 CNV in CAISMOV24), most of them were related to CNVs already present in the primary malignant cells. CAISMOV24 harbored KRAS mutation with wild type TP53, therefore it is characterized as low-grade serous carcinoma. Conclusions: The functional performance of NK cells was distinct between EOC cell-free ascites and ascites with EOC cells. Ascites with EOC cells displayed an immunosuppressive microenvironment, with high levels of T-reg lymphocytes, suppression of NK cells activating receptors and inefficient response to IL-2, measure through degranulation. The cell line CAISMOV24 was characterized as a low-grade serous ovarian carcinoma, similar to its primary malignant cells. Our study corroborates with the idea that genomic alterations, depicted by CNVs, can be used for subtyping ovarian carcinoma (AU)

• FAPESP's process: 14/07401-3 - Interaction of T-reg and NK lymphocytes from ovarian cancer patients during cytotoxic activity against ovarian neoplasia
• Grantee: Rodrigo Fernandes da Silva
• Support Opportunities: Scholarships in Brazil - Doctorate