Organotypic Triple-Negative Breast Cancer Model

The organotypic tumor model was formed in two robotic micropatterning steps using an aqueous two-phase system (ATPS) technology with 5.0%(w/v) polyethylene glycol and 6.4%(w/v) dextran (17 (link),18 (link)). First, 7.5×10³ TNBC cells were microprinted in a nanodrop to form a spheroid in each well of a 384-well plate. Then, a drop containing 20 μl ice-cold rat tail type I collagen solution (Corning) and 1.5×10⁴ suspended fibroblasts was dispensed into each well. The 384-well plate was incubated at 37°C for 30 min to form the tumor model with fibroblasts dispersed in a collagen gel containing a mass of TNBC cells. For confocal imaging, the tumor models were formed in a glass bottom 384-well plate (MatTek, Cat. No. PBK384G). Images were captured with a 10X objective on days 1 and 5 of culture (Nikon A1). TNBC cells without GFP were stained with Calcein AM (Thermo Fisher Scientific). Z-projected images were constructed from images of samples acquired with a z-spacing of 20 μm. NIS software was used for image acquisition and Fiji (ImageJ, NIH) was used for image analysis and 3D reconstruction. The total pixel area of TNBC cells when cultured with CAF-1 or CAF-2 was normalized to the pixel area of TNBC cells when cultured with HMF. Unlabeled collagen fibers were imaged using confocal reflectance microscopy.