(F-205) Therapeutic potency testing of drug-loaded microparticles against glioblastoma using a perfusable tumor-on-chip platform

Introduction: Glioblastoma multiforme (GBM) is a primary brain tumor with a low survival rate of 12-15 months post-diagnosis and treatment. The current standard of care (SOC) treatments for GBM involves maximal surgical resection followed by radiotherapy and chemotherapy, which is ineffective and does not provide any survival benefit to patients. The poor prognosis of GBM patients is also attributed to the development of therapeutic resistance owing to the presence of a highly specialized tumor microenvironment (TME) consisting of a heterogeneous blood tumor barrier (BTB) and a highly modified extracellular matrix (ECM), which acts as a molecular and physical barrier to tumor-targeting therapeutics. Current in vitro drug testing platforms fail to replicate the physiologically relevant GBM TME and largely overlook the vital role of the TME in promoting therapeutic resistance and in aiding GBM progression. In order to address this technological gap, we designed a novel GBM-on-chip platform consisting of a perfusable vascularized flow channel, barriers separating the flow channel from tumor reservoirs, and tumor reservoirs that can be used to immobilize tumor organoids or tissue in a TME-relevant ECM matrix. We hypothesized that the physiologically relevant GBM-on-chip platform technology can be used to determine accurate therapeutic dosing and monitor the efficacy of tumor-targeting SOC and combination therapies.


Method: We synthesized poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) loaded with Surfen, a sulfated glycosaminoglycan inhibitor - bis-4-aminoquinoline. This facilitates the targeted, sustained release of drug combined with a chemotherapeutic drug, temozolomide (TMZ), provides multi-modal activity against GBM cells. For drug efficacy studies, patient-derived GBM cells with distinct mutations were cultured in growth factor-supplemented Neurobasal-A media at 37°C and 5% CO2. After passaging, GBM spheroids were cultured in the same media at 2 × 104 cells/well density in ultra-low attachment 96-well plates. Microfluidic devices were fabricated using PDMS with a 10:1 ratio of base to crosslinker with a microfabricated mold. We tested the efficacy of TMZ at 500, 1000, and 2000 µM and Surfen at 20, 40, and 80µM, and a combination of both at selected dosages in GBM-on-chip or 96 well plates two days after seeding spheroids. Afterward, combination therapy was tested against patient-derived GBM neurospheres using drug-loaded PLGA MPs that were added to the tumor site, and media containing TMZ that was circulated at a flow rate of 10µl/min. Cell viability, proliferation, apoptosis and cell cycle progression, and tumor growth were assessed using flow cytometry and imaging techniques. We also characterized the biophysical features of optimized microcarriers, including their size, shape, surface properties, and release kinetics of TMZ and Surfen.


Results: Drug dose-response studies using TMZ, Surfen, and combination (TMZ 1000µM/Surfen 20µM) demonstrated a significant reduction in GBM cell viability, proliferation, and invasion in a dose-dependent manner. PLGA microparticles synthesized using a double emulsion technique demonstrated an average size of 1 µm, zeta -36 mV. Scanning electron microscopy (SEM) showed the microparticles had a smooth surface and a spherical geometry. The encapsulation efficiency and release profile of drugs from microparticles were investigated and showed an initial burst followed by a continuous drug release of Surfen at the rate of maximum 20% per day for up to 7 days in vitro. The drug-loaded MPs also induced cell cycle arrest and apoptosis in GBM spheroids. Cytotoxicity assays suggested that GBM spheroids were more sensitive to the combination of TMZ and Surfen compared to either agent alone when delivered from PLGA polymeric carrier. Our findings suggest that combination therapy using TMZ and Surfen may be a promising strategy for treating GBM patients.


Conclusion: In conclusion, compartmentalizing of multiple relevant cell types in the GBM-on-chip mimics the TME and provides a physiologically relevant platform for investigating the antitumor potency of drugs and cellular immunotherapies under dynamic conditions and over a longer time course than feasible using static well plate assays. We also demonstrated that TMZ and Surfen can effectively inhibit GBM cell growth and invasion in vitro while having a synergistic effect when used together. Our future studies will develop strategies to formulate local delivery and evaluate the effects of co-administering Surfen/TMZ better in vitro and in vivo.