Tumor Spheroid-Neutrophil Co-Culture Assay

To evaluate neutrophil recruitment toward cancer cells in real time, a co-culture model system was developed ( Figure 5A ). Wells of a 24 well glass-bottom plate (P24-1.5H-N, Cellvis) were coated with 200 µg/ml fibronectin (F1141, Sigma-Aldrich) for at least 1 hr at 37°C. Approximately 20 spheroids resuspended in 250 µl fresh media ( Table 1 ) supplemented with reduced serum (1%) were plated in each well. Spheroids were allowed to attach to the plate for about 4 hrs in the incubator at 37°C/5% CO₂. Towards the end of incubation, neutrophils were primed with 5 ng/ml GM-CSF for 30 mins. 5x10⁴ or 1x10⁵ primed neutrophils were seeded to each well in 250 µl of 1% serum media. Multiple spheroids in each well were imaged for 24 hrs using an environmental controlled Zeiss Colibri microscope at 10x magnification. To analyze the migration of the neutrophils, the paths of at least 40 neutrophils per video, spaced out both spatially and temporally, were manually tracked using the Manual Tracking plugin in ImageJ software. The position data acquired from the tracking were used in a custom MATLAB script to quantify X-FMI.

TNBC spheroids actively recruit neutrophils. (A) Cartoon depicting the co-culture migration system used to assess the chemotaxis of neutrophils towards tumor spheroids in fibronectin coated glass-bottom plate. (B, C) Representative images of a neutrophil-spheroid co-culture migration assay at 1, 10, and 20 hrs with M4 spheroid (B) or BT474 spheroid (C) at the center of the field of view. Small, dark cells are neutrophils from the same donor. Representative tracks of neutrophils are on the right. (D) Graph depicting the X-FMI of neutrophils migrating towards M4 or BT474 spheroids. Each bar represents mean ± SEM from N= 3 (BT474 spheroid) or 4 (M4 spheroid) independent donors with each dot representing the average X-FMI for all neutrophils tracked per spheroid. An average of 45 neutrophils were tracked per spheroid. ****P < 0.0001 when compared with BT474 (unpaired t-test).