Lipid droplets and perilipins in canine osteosarcoma. Investigations on tumor tissue, 2D and 3D cell culture models

Abstract

Lipid droplets were identified as important players in biological processes of various tumor types. With emphasis on lipid droplet-coating proteins (perilipins, PLINs), this study intended to shed light on the presence and formation of lipid droplets in canine osteosarcoma. For this purpose, canine osteosarcoma tissue samples (n = 11) were analyzed via immunohistochemistry and electron microscopy for lipid droplets and lipid droplet-coating proteins (PLINs). Additionally, we used the canine osteosarcoma cell lines D-17 and COS4288 in 2D monolayer and 3D spheroid (cultivated for 7, 14, and 21 days) in vitro models, and further analyzed the samples by means of histochemistry, immunofluorescence, molecular biological techniques (RT-qPCR, Western Blot) and electron microscopical imaging. Lipid droplets, PLIN2, and PLIN3 were detected in osteosarcoma tissue samples as well as in 2D and 3D cultivated D-17 and COS4288 cells. In spheroids, specific distribution patterns of lipid droplets and perilipins were identified, taking into consideration cell line specific zonal apportionment. Upon external lipid supplementation (oleic acid), a rise of lipid droplet amount accompanied with an increase of PLIN2 expression was observed. Detailed electron microscopical analyzes revealed that lipid droplet sizes in tumor tissue were comparable to that of 3D spheroid models. Moreover, the biggest lipid droplets were found in the central zone of the spheroids at all sampling time-points, reaching their maximum size at 21 days. Thus, the 3D spheroids can be considered as a relevant in vitro model for further studies focusing on lipid droplets biology and function in osteosarcoma.

Isolation and Characterization of Novel Canine Osteosarcoma Cell Lines from Chemotherapy-Naïve Patients

“…Cells were cultured for at least 20 passages before entering the characterization process, except for COS4074 cells, where the characterization started at passage 17, representing 5–6 month of continuous COS4074 cell cultivation. The isolation of COS4288 cells was carried out in a similar manner, as recently described in [ 26 ].…”

“…The soft agar colony formation assay was performed as described [ 26 ]. Colony formation was evaluated after 21 days for COS3600B, COS4288, and D-17 and after 56 days for COS3600 and COS4074 cells.…”

“…Spheroids were grown as described [ 26 ]. In short, 2 × 10 4 of D-17 cells or 4 × 10 4 of COS4288 cells were seeded per well of a 96-well low attachment plate (96 w Brand plates U inert Grade clear, Brand GmbH, Wertheim, Germany) and cultivated for up to 21 days.…”

“…Cells for immunohistochemical analyses were grown on 4-well glass chamber slides (Lab-Tek II Chamber Slide System, ThermoFisher Scientific, Waltham, MA, USA). Immunofluorescent detection of cytokeratin and vimentin as well as immunodetection of alkaline phosphatase (ALPL) and karyopherin α2 (KPNA2) was carried out as described [ 26 ].…”

Morphologic, phenotypic, and genotypic similarities between primary tumors and corresponding 3D cultures grown in a repeatable system

“…Canine cancer cell have been cultured most commonly as 2D cultures obtained from several types of neoplasms, including soft and hard tissue sarcoma [11][12][13][14] and different histotypes of carcinoma [15][16][17] to demonstrate feasibility, obtain abundant neoplastic cell material for genetic studies and for therapy e cacy assessment. For osteosarcoma, lymphoma [18,19] and mammary carcinoma, [17] 3D cultures systems have been recently used to study speci c target therapies and demonstrate the importance of the tumor speci c stromal microenvironment. These studies have demonstrated the better performances and the essential role of 3D culture systems for the understanding of treatment response and the emergence of chemo-resistant clones.…”