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Three-Dimensional Cell Culture Based on Magnetic Fields to Assemble Low-Grade Ovarian Carcinoma Cell Aggregates Containing Lymphocytes.

ABSTRACT: There is a limited number of established ovarian cancer cell lines matching the low-grade serous histotype available for research purposes. Three-dimensional (3D) culture systems provide in vitro models with better tissue-like characteristics than two-dimensional (2D) systems. The goal in the study was to characterize the growth of a given low-grade serous ovarian carcinoma cell line in a 3D culture system conducted in a magnetic field. Moreover, the culture system was evaluated in respect to the assembly of malignant cell aggregates containing lymphocytes. CAISMOV24 cell line alone or mixed with human peripheral blood mononuclear cells (PBMC) were cultured using a commercially available 3D culture system designed for 24 well plates. Resulting cell aggregates revealed the intrinsic capacity of CAISMOV24 cells to assemble structures morphologically defined as papillary, and reflected molecular characteristics usually found in ovarian carcinomas. The contents of lymphocytes into co-cultured cell aggregates were significantly higher (p < 0.05) when NanoShuttle-conjugated PBMC were employed compared with non-conjugated PBMC. Moreover, lymphocyte subsets NK, T-CD4, T-CD8 and T-regulatory were successfully retrieved from co-cultured cell aggregates at 72h. Thus, the culture system allowed CAISMOV24 cell line to develop papillary-like cell aggregates containing lymphocytes.

SUBMITTER: Natania de Souza-Araujo C

PROVIDER: S-EPMC7140502 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Three-Dimensional Cell Culture Based on Magnetic Fields to Assemble Low-Grade Ovarian Carcinoma Cell Aggregates Containing Lymphocytes.

Natânia de Souza-Araújo Caroline C Rodrigues Tonetti Cláudia C Cardoso Marcella Regina MR Lucci de Angelo Andrade Liliana Aparecida LA Fernandes da Silva Rodrigo R Romani Fernandes Luís Gustavo LG Guimarães Fernando F

Cells 20200306 3

There is a limited number of established ovarian cancer cell lines matching the low-grade serous histotype available for research purposes. Three-dimensional (3D) culture systems provide in vitro models with better tissue-like characteristics than two-dimensional (2D) systems. The goal in the study was to characterize the growth of a given low-grade serous ovarian carcinoma cell line in a 3D culture system conducted in a magnetic field. Moreover, the culture system was evaluated in respect to th ...[more]

PMID: 32155738

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Project description:Background3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts.Methodology/principal findingsWe compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medium devoid of animal-derived components, using breast cancer cell lines representative of the different molecular subtypes of breast cancer, cultured alone or with human mammary fibroblasts with a view to developing matrix-free humanised systems. 3D collagen I culture supported the growth of a range of breast cancer cell lines. By modifying the composition of Fibrolife® to epiFL, matrix-free cell culture was possible. During sequential transfer to epiFL breast cancer cells gradually detached from the flask, growing progressively as spheroids. Phenotype was stable and reversible with cells remaining actively proliferating and easily accessible throughout culture. They could also be revived from frozen stocks. To achieve co-culture with fibroblasts in epiFL required use of low attachment culture vessels instead of standard plastic as fibroblasts remained adherent in epiFL. Here, cancer cell spheroids were allowed to form before adding fibroblasts. Immunohistochemical examination showed fibroblasts scattered throughout the epithelial spheroid, not dissimilar to the relationship of tumour stroma in human breast cancer.ConclusionsBecause of its ease of handling, matrix-free 3D cell culture may be a useful model to study the influence of fibroblasts on breast cancer epithelial cells with use of epiFL culture medium taking this a step further towards a fully humanised 3D model. This methodology could be applied to other types of cancer cell lines, making this a versatile technique for cancer researchers wishing to use in vitro systems that better reflect cancer in vivo.

Project description:Importance:Cytotoxic CD8+ tumor-infiltrating lymphocytes (TILs) participate in immune control of epithelial ovarian cancer; however, little is known about prognostic patterns of CD8+ TILs by histotype and in relation to other clinical factors. Objective:To define the prognostic role of CD8+ TILs in epithelial ovarian cancer. Design, Setting, and Participants:This was a multicenter observational, prospective survival cohort study of the Ovarian Tumor Tissue Analysis Consortium. More than 5500 patients, including 3196 with high-grade serous ovarian carcinomas (HGSOCs), were followed prospectively for over 24?650 person-years. Exposures:Following immunohistochemical analysis, CD8+ TILs were identified within the epithelial components of tumor islets. Patients were grouped based on the estimated number of CD8+ TILs per high-powered field: negative (none), low (1-2), moderate (3-19), and high (?20). CD8+ TILs in a subset of patients were also assessed in a quantitative, uncategorized manner, and the functional form of associations with survival was assessed using penalized B-splines. Main Outcomes and Measures:Overall survival time. Results:The final sample included 5577 women; mean age at diagnosis was 58.4 years (median, 58.2 years). Among the 5 major invasive histotypes, HGSOCs showed the most infiltration. CD8+ TILs in HGSOCs were significantly associated with longer overall survival; median survival was 2.8 years for patients with no CD8+ TILs and 3.0 years, 3.8 years, and 5.1 years for patients with low, moderate, or high levels of CD8+ TILs, respectively (P value for trend?=?4.2?×?10?16). A survival benefit was also observed among women with endometrioid and mucinous carcinomas, but not for those with the other histotypes. Among HGSOCs, CD8+ TILs were favorable regardless of extent of residual disease following cytoreduction, known standard treatment, and germline BRCA1 pathogenic mutation, but were not prognostic for BRCA2 mutation carriers. Evaluation of uncategorized CD8+ TIL counts showed a near-log-linear functional form. Conclusions and Relevance:This study demonstrates the histotype-specific nature of immune infiltration and provides definitive evidence for a dose-response relationship between CD8+ TILs and HGSOC survival. That the extent of infiltration is prognostic, not merely its presence or absence, suggests that understanding factors that drive infiltration will be the key to unraveling outcome heterogeneity in this cancer.

Dataset Information

Three-Dimensional Cell Culture Based on Magnetic Fields to Assemble Low-Grade Ovarian Carcinoma Cell Aggregates Containing Lymphocytes.

Publications

Three-Dimensional Cell Culture Based on Magnetic Fields to Assemble Low-Grade Ovarian Carcinoma Cell Aggregates Containing Lymphocytes.

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