High-throughput monitoring of cancer cell/fibroblast cocultures, and their interaction with TNF-α
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High-throughput monitoring of cancer cell/fibroblast cocultures, and their interaction with TNF-α Marc van Vijven, PhD; Inge Thijssen-van Loosdregt, PhD CytoSMART® Technologies B.V., Eindhoven, The Netherlands Introduction Live-cell imaging enables researchers to determine not only whether, but also when and how certain cellular events occur in culture. When compared to brightfield-only imaging, fluorescence live-cell imaging multiplies the number of readouts– and consequently the obtained information from one experiment – with the number of fluorescence channels. In cellular co-cultures, fluorescence imaging facilitates distinction of the cell types, when cell types are assigned specific fluorescent labels. Quantitative and qualitative read-outs per cell type are then based on the respective fluorescent labels [1]. Fig. 1. The CytoSMART® Omni FL Co-culture setups are commonly applied in cancer research, since tumors and their environment involve many different a stage-top incubation box. However, regulation of the culture cell types: e.g., immune cells aim for elimination of the cancer conditions in the incubation box – usually 37° and 5% CO2 – is C cells [2], endothelial cells are stimulated to vascularize a more sensitive to variations compared to a dedicated incubator tumor [3], and fibroblasts can either promote or inhibit tumor [10, 11]. Besides that, motorized movement of the sample stage growth [4-6]. Two types of fibroblasts have been found in is necessary to image all wells in a culture plate used for high- tumor cell microenvironments. On the one hand, cancer- throughput experiments. This increases not only the total cost associated fibroblasts (CAFs) have tumor-promoting and of the setup, but medium flow resulting from sample movement immunosuppressive activity. These fibroblasts get activated by can also alter cell behavior and consequently the experiment tumor necrosis factor-α (TNF-α), which is secreted by cancer cells [6-9]. On the other hand, normal-associated fibroblasts (NAFs) secrete TNF-α themselves, thereby inhibiting tumor The CytoSMART® Omni FL (Fig. 1) is a high-throughput growth [6, 7]. The fact that TNF-α can perform both conflicting fluorescence live-cell imager that could overcome these issues. functions relates to the TNF receptors expressed by tumor The device can be placed on a single shelf of a regular incubator, cells, TNFR1 and TNFR2. These receptors influence each other’s and therefore enables two-channel fluorescence live-cell downstream effects, and thereby together orchestrate TNF-α imaging (green, red) of a well-plate in a constant and optimal functionality in a tumor microenvironment-dependent manner culture environment. Since the fluorescence module and camera are moving while the plate remains stationary, all wells in a culture plate can be imaged without medium flow resulting The confluence per cell type can provide insight into interactions from sample movement disturbing the cells. between cancer cells and different fibroblast types: CAFs supporting cancer cell proliferation or NAFs competing with and In this proof-of-concept study, we determined the effect of both eliminating tumor cells [6]. Fundamental knowledge involving the seeding ratio and TNF-α concentration on proliferation of these cellular interactions can be relevant in cancer research, a cancer cell line and fibroblasts in co-culture. The CytoSMART® where potential clinical therapies could be based on insight in Omni FL and corresponding Cloud-based image analysis conditions in which cancer cells are eliminated by surrounding algorithm for brightfield and fluorescent channel confluence fibroblasts. To study interaction effects of cancer cell/fibroblast were applied. With these tools, the kinetic confluence profiles of co-cultures and TNF-α, all possible combinations of conditions fluorescently labeled HeLa cells (cervical carcinoma cell line) and per experimental variable have to be covered. However, this 3T3 cells (fibroblasts) in co-cultures with six different seeding two-factor experiment design requires an imaging system that ratios and four different TNF-α concentrations were determined. enables high-throughput fluorescence live-cell imaging of the This high-throughput experiment provided fundamental insight co-cultures over time. Currently, fluorescence live-cell imaging is into TNF-α-dependent interactions between the cell types, predominantly performed using a fluorescence microscope with

High-throughput monitoring of cancer cell/fibroblast cocultures, and their interaction with TNF-α Marc van Vijven, PhD; Inge Thijssen-van Loosdregt, PhD CytoSMART® Technologies B.V., Eindhoven, The Netherlands Materials and methods tGFP-labeled HeLa cells (Innoprot P20107; green fluorescent) Co-cultures at each of the cell ratios were exposed to various and non-fluorescent 3T3 fibroblasts (ATCC® CL-173™) were concentrations of TNF-α (Merck H8916): 0 ng/ml, 0.2 ng/ml, separately cultured to sub-confluency in DMEM (Gibco) 2 ng/ml, and 20 ng/ml [13, 14]. All conditions were monitored...

High-throughput monitoring of cancer cell/fibroblast cocultures, and their interaction with TNF-α Marc van Vijven, PhD; Inge Thijssen-van Loosdregt, PhD CytoSMART® Technologies B.V., Eindhoven, The Netherlands Fig. 3. Confluence quantifications per cell type over time for HeLa : 3T3 co-cultures, corresponding to the various seeding ratios and TNF-α concentrations. All conditions were monitored with the CytoSMART® Omni FL over 48 h with a 1 h imaging interval and 3 images per well. The confluence per channel was assessed in the CytoSMART® Cloud, and averaged over the 3 images per well. All...

High-throughput monitoring of cancer cell/fibroblast cocultures, and their interaction with TNF-α Marc van Vijven, PhD; Inge Thijssen-van Loosdregt, PhD CytoSMART® Technologies B.V., Eindhoven, The Netherlands The confluence quantifications over time indicated competition suppressing properties of NAFs became stronger with higher between the HeLa cells and the 3T3 cells. However, which of the environmental TNF-α concentrations. The Omni FL enabled cell types was eliminated was dependent on the initial ratio. In monitoring of this multi-factor experimental setup, thereby previous research,...