Development of a Microfluidic Organ-on-a-Chip Platform for High-Throughput Screening of Anti-Cancer Immunotherapies Targeting PD-1/PD-L1 Pathway

Immune checkpoint blockades targeting the programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) axis have fundamentally reshaped modern clinical oncology; however, conventional two-dimensional cell cultures and animal models frequently fail to recapitulate the complex human tumor microenvironment (TME) or afford high-throughput evaluation. Here, we present the design, fabrication, and analytical validation of an automated, 96-unit multiplexed microfluidic organ-on-a-chip platform engineered specifically for high-throughput screening of PD-1/PD-L1 immunotherapeutic candidates. The platform incorporates biomimetic three-dimensional tumor spheroids embedded within an extracellular matrix hydrogel, flanked by perfusable endothelialized vascular conduits, allowing physiological extravasation and migration of primary human CD8+ cytotoxic T lymphocytes under controlled interstitial flow. Utilizing real-time high-content fluorescence imaging and automated image processing, we quantified dynamic immune cell infiltration, target engagement, and localized tumor-cell cytolysis upon treatment with clinical therapeutic antibodies and synthetic small-molecule inhibitors. Treatment with PD-1/PD-L1 antagonists elicited a statistically significant dose-dependent increase in T-cell extravasation velocity, a 3.8-fold elevation in intratumoral granzyme B release, and a marked induction of cleaved caspase-3 in human non-small cell lung cancer (A549) spheroids within 48 hours. Parallel on-chip effluent sampling corroborated localized upregulation of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). This microengineered platform bridges the gap between conventional static assays and in vivo preclinical trials, providing an efficient, standardized, and scalable framework for accelerated immunotherapy discovery and personalized medicine profiling.