A major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components.
Reconstitution of an HER2+ Tumor Ecosystem On-Chip, Including CAFs and Immune Cells
We designed a microfluidic device for cell co-cultures (called a chip for short), based on published works (Chen et al., 2017, Lucarini et al., 2017, Parlato et al., 2017, Zervantonakis et al., 2012) (Figure 1A). The chips were microfabricated by soft lithography using PDMS (polydimethylsiloxane), a silicone rubber that is biocompatible, gas permeable, and transparent. The chip design consisted of 5 parallel microchambers (500- to 1000-μm wide, 150- to 200-μm high), separated by regularly spaced micropillars that allow the confinement of hydrogels by means of a balance between surface tension and capillary forces. The various cell types were positioned inside the chips, mimicking their original in vivo architecture in tumors.
Cancer cells (the HER2+ breast cancer BT474 cell line), cancer-associated fibroblasts (the breast CAF cell line Hs578T), and immune cells (PBMCs [peripheral blood mononuclear cells] from healthy donors) were embedded into 3D biomimetic hydrogels (made of collagen type I at 2.3 mg/mL, the major component of the extracellular matrix [ECM]), inside the 2 inner lateral chambers. To compare conditions with and without CAFs within the same chip, the left gel chamber was without CAFs, while the right gel chamber was with CAFs. The 2 outer lateral chambers were used as medium reservoirs. This work was focused on the effects of CAFs and immune cells; however, endothelial cells were always included since in vivo they contribute to shaping the biochemical environment by secreting a variety of cytokines and other soluble factors ( Buchanan et al., 2012, Lee et al., 2015 ). Endothelial cells (primary human umbilical vein endothelial cells [HUVECs]) were grown as 2D monolayers in the central chamber, as previously reported (Zervantonakis et al., 2012). For simplicity, the majority of co-cultures and observations were performed without adding perfusion in this vessel compartment. Considering that 3D cell co-cultures were achieved in microfluidics devices and that the literature extensively uses the organ-on-chip terminology even for systems without perfusion, we adopted the tumor-on-chip terminology for our approach.
These tumors-on-chip were visualized by high-content video-microscopy (multi-positioning, multi-colors, 2- to 120-min time intervals) for 4–5 days (Video S1). To discriminate cancer cells from CAFs, the fibroblasts were pre-stained in red with a live dye (CellTrace Yellow reagent) (Video S2). In addition, to quantitatively describe the tumor ecosystem, we implemented appropriate methods to measure on-chip proliferation, apoptotic death, and cell-cell interactions (Figures 1B and 1C). To monitor proliferation, nuclei were live stained using a far-red nuclear dye (SiR-DNA), which allows for the identification and counting of mitotic events (Video S3). To monitor cell death, apoptotic cells were identified using a green probe detecting caspase 3 and 7 activity (CellEvent caspase-3/7) (Video S4). Cells undergoing apoptosis, in addition to emitting green fluorescence, also showed a change in morphology. The overall observation of hundreds of videos revealed extremely rich and complex cell-cell interactions involving immune cells (Video S5). Using the ad hoc-designed CellHunter automated method (
Biselli et al., 2017, Parlato et al., 2017), we tracked the cell dynamics within the co-cultures and measured the interaction times with each immune cell for each cancer cell (Figure 1C), thus providing a high-content description of the cancer-immune cell interactions inside the reconstituted tumor ecosystem. The relative positioning and morphologies of the different cell populations were investigated by live snapshot confocal microscopy; cancer cells, immune cells, and fibroblasts are distributed along the z dimension of the collagen gel, and several cell-cell contacts occur in this true 3D ecosystem. The endothelial cells create vertical barriers, although not continuous ones, at the interfaces between the endothelium channel and the collagen gels (Figure 2).
Authors: Marie Nguyen, Adele De Ninno, Arianna Mencattini, Fanny Mermet-Meillon, Giulia Fornabaio, Sophia S. Evans, Mélissande Cossutta, Yasmine Khira, Weijing Han, Philémon Sirven, Floriane Pelon, Davide Di Giuseppe, Francesca Romana Bertani, Annamaria Gerardino, Ayako Yamada, Stéphanie Descroix, Vassili Soumelis, Fatima Mechta-Grigoriou, Gérard Zalcman, Jacques Camonis, Eugenio Martinelli, Luca Businaro, Maria Carla Parrini