Poster S17 - ASTEROIDS- tumor microenvironment modeling with novel fluidic device

Introduction: Historic translational disparity of basic medical science to clinical application in oncology has supported investment in developing more human physiologically relevant pre-clinical models. To this end, we here propose a new approach combining three-dimensional cancer cell culture with a microfluidic system supporting the culture of stromal cells in a dynamic environment. Therefore, our group designed and tested a novel organ-on-chip called Apparatus to Simulate Tumor and Reproduce Organs in an Interactive and Dynamic System (ASTEROIDS). As a proof-of-concept, ASTEROIDS was bioengineered to recapitulate aspects of the lung tumor microenvironment (TME) by using hydrogel-embedded A549 lung cancer spheroids cultured in central wells surrounded by two parallel perfused chambers seeded with lung endothelial (HULEC-5a) and fibroblast (IMR-90) cells. ASTEROIDS design has been specifically engineered to provide maximum versatility for the end user by supporting multi-applications such as direct imaging, microplate reader utilities, and ergonomic assembly/disassembly for maximum tissue recovery.

Materials and

Methods: Central piece and parallel chambers were produced by injection molding of optically clear polycarbonate. Chambers were separated from the central piece by an 8 µm pore-size polycarbonate film, before being assembled by clamping using silicon gaskets. Collagen-coated polycarbonate films were then seeded with HULEC-5a and IMR90 before the loading of pre-formed A549 spheroids embedded in basement membrane matrix in the central wells. Side chambers with HULEC-5a and IMR90, mimicking vascular and connective tissue respectively, were then perfused at a flow rate of 60 µL/min for 7 days. Direct imaging, immunofluorescence (IF) staining and fluorescent dextran assay were then performed to assess spheroid growth, HULEC-5a and IMR90 viability, and endothelial barrier function. Transcriptomic profiling of HULEC-5a cells cultured in presence or absence of A549 spheroids was also performed by qRT-PCR to investigate intercellular communication. Peripheral blood mononuclear cells (PBMCs) were then isolated from healthy blood donors using density gradient centrifugation. These PBMCs were injected in the vascular chamber of the ASTEROIDS previously activated with TNFα to investigate PBMCs interaction with endothelial cells under physiological flow. Finally, clinically relevant radiation doses were delivered to the device. IF and qRT-PCR were then performed on both cancer and non-cancer cells to investigate cellular response to radiation in the ASTEROIDS.

Results, Conclusions, and Discussions: After 7 days of culture, data showed that the area of A549 spheroids increased by 2x. Live/dead IF characterization showed both HULECs-5a and IMR90 cells were alive. In addition, HULEC-5a displayed barrier maturation by VE-Cadherin structuring and decreased apparent permeability compared to the unseeded polycarbonate films. In addition, HULEC-5a transcriptomic profiles showed an increase of Myc-target genes expression in presence of A549 spheroids as previously observed by in-vivo lung tumor biopsies vs. normal lung tissue. The injection of PBMCs showed a significant increase of CD45-positive cells on HULECs-5a membrane after TNFα activation and in presence of A549 spheroids. Finally, irradiation at 0, 8, and 24 Gy revealed a dose-dependent rise in γH2AX/53BP1 foci in HULEC-5a and IMR90 cells. In addition, endothelial barrier disruption was observed by decreased VE-Cadherin expression and increased permeability while IMR90 entered radiation-induced senescence. The A549 spheroids also showed significant growth inhibition and immunogenic cell death was observed only after 24 Gy by live-imaging and Calreticulin IF staining, respectively. Current investigations are assessing if ASTEROIDS perfusion of PBMCs could reproduce the tumor response to SBRT as recently reported by in vivo studies using functional and transcriptomics analyses and showing alterations of immune component and extracellular matrix.

This work showed that ASTEROIDS can sustain the co-culture up to 7 days and that the cells even demonstrated establishment and maintenance of key tissue features such as endothelial barrier function or tumor pathophysiological gradients. In addition, our results demonstrated that an intercellular communication occurred within the different ASTEROIDS compartments as shown by the alteration of the HULEC-5a transcriptomics profile and PBMCs recruitment in presence of A549 cells. Finally, the ASTEROIDS could sustain irradiation and cells have been shown to be responsive to this stimulus, suggesting that, overall, the platform provides a physiologically relevant human in vitro model to study TME response to radiation.

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