In vitro screening methods of novel immune checkpoint inhibitors related to T cell infiltration and anti-PD-1 resistance.

Immune checkpoint blockade-based cancer immunotherapy is an effective tool for cancer treatment. PD-1/PD-L1 blockade, however, is limited by a low response rate and adaptive resistance. A growing body of studies has shown that the high stromal content dense with extracellular matrix plays a significant role in immune checkpoint blockade resistance as well as T cell exclusion. In addition to physically obstructing immune cell infiltration, the extracellular matrix (ECM) may also interact with T cell receptors to indirectly impair their effector function and lead to anti-PD-1 resistance. Anti-PD-1 resistance may thus be overcome by rupturing the physical barrier related negative immune regulation, which may improve T cell infiltration and the efficacy of cancer immunotherapy. Here, we offer two straightforward methods based on flow cytometry and confocal microscopy to evaluate the effectiveness of an inhibitor targeting the novel "stromal checkpoint" DDR1/collagen, which aims to facilitate T cell migration and infiltration of tumor spheres by overcoming collagen barriers. With minor variations, the same method can be easily modified to test the inhibitors that target other immune checkpoints, and the extracellular matrix-associated drug targets that mediate anti-PD-1 resistance.