X-ray on chip: Quantifying therapeutic synergies between radiotherapy and anticancer drugs using soft tissue sarcoma tumor spheroids.

PURPOSE: Radioresistance, tumor microenvironment, and normal tissue toxicity from radiation limit the efficacy of radiotherapy in treating cancers. These challenges can be tackled by the discovery of new radiosensitizing and radioprotecting agents aimed at increasing the therapeutic efficacy of radiotherapy. The goal of this work was to develop a miniaturized microfluidic platform for the discovery of drugs that could be used in combination with radiotherapy. The microfluidic system will allow the toxicity testing of cancer spheroids to different combinations of radiotherapy and molecular agents. MATERIALS AND METHODS: An orthovoltage-based technique was used to expose the devices to multiple X-ray radiation doses simultaneously. Radiation dose-dependent DNA double-strand breaks in soft tissue sarcoma (STS) spheroids were quantified using comet assays. Analysis of proliferative death using clonogenic assays was also performed, and synergy between treatments with Talazoparib, Pazopanib, AZD7762, and radiotherapy was quantified using dedicated statistical tests. RESULTS: The developed microfluidic system with simple magnetic valves was capable of growing 336 homogeneous STS spheroids. The irradiation of the microfluidic system with an orthovoltage-based technique enabled the screening of sixteen drug-radiotherapy combinations with minimal reagent consumption. Using this framework, we predicted a therapeutic synergy between a novel anticancer drug Talazoparib and radiotherapy for STS. No synergy was found between RT and either Pazopanib or AZD7762, as the combinations were found to be additive. CONCLUSION: This methodology lays the basis for the systemic search for molecular agent/radiotherapy synergies among preexisting pharmaceutical compounds libraries, in the hope to identify failed drug candidates in monotherapy that, in the presence of radiotherapy, would make it through clinical trials.

Engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics.

Tunable bioprinting materials are capable of creating a broad spectrum of physiological mimicking 3D models enabling in vitro studies that more accurately resemble in vivo conditions. Tailoring the material properties of the bioink such that it achieves both bioprintability and biomimicry remains a key challenge. Here we report the development of engineered composite hydrogels consisting of gelatin and alginate components. The composite gels are demonstrated as a cell-laden bioink to build 3D bioprinted in vitro breast tumor models. The initial mechanical characteristics of each composite hydrogel are correlated to cell proliferation rates and cell spheroid morphology spanning month long culture conditions. MDA-MB-231 breast cancer cells show gel formulation-dependency on the rates and frequency of self-assembly into multicellular tumor spheroids (MCTS). Hydrogel compositions comprised of decreasing alginate concentrations, and increasing gelatin concentrations, result in gels that are mechanically soft and contain a greater number of cell-adhesion moieties driving the development of large MCTS; conversely gels containing increasing alginate, and decreasing gelatin concentrations are mechanically stiffer, with fewer cell-adhesion moieties present in the composite gels yielding smaller and less viable MCTS. These composite hydrogels can be used in the biofabrication of tunable in vitro systems that mimic both the mechanical and biochemical properties of the native tumor stroma.

On-chip combined radiotherapy and chemotherapy testing on soft-tissue sarcoma spheroids to study cell death using flow cytometry and clonogenic assay.

Radiotherapy (RT) and chemotherapy (CT) are the major therapeutics to treat cancer patients. Conventional in vitro 2D models are insufficient to study the combined effects of RT and CT towards optimized dose selection or drug screening. Soft-tissue sarcomas (STS) are rare cancers with profound social impacts as they affect patients of all ages. We developed a microfluidic device to form and culture STS spheroids to study the combined cytotoxicities of RT and CT. Uniformly-sized spheroids of two different cell lines, STS 93 and STS 117, were formed in the device. RT doses of 0.5 Gy, 2 Gy, and 8 Gy were used in combination with CT, doxorubicin at 2 µM and 20 µM. The spheroids culture chambers within the device were arranged in a 3 × 5 matrix form. The device was made "peelable", which enabled us to collect spheroids from each treatment condition separately. Collected spheroids were dissociated into single cells and evaluated using flow cytometry and clonogenic assays. Through this workflow, we observed that STS 93 spheroids treated with doxorubicin die through apoptosis, whereas RT induced death through other pathways. Spheroids from the p53 mutant STS 117 cell line were more resistant to RT and doxorubicin. The developed device could be used for the discovery of new drugs and RT synergies.