Study of immune regulatory receptor expression upon combination of chemotherapy and immune checkpoint blockade in a murine lung cancer model

Abstract

Background: Blockade of inhibitory immune checkpoints namely the programmed cell death protein 1 (PD-1) and the PD-1 ligand (PD-L1) achieved remarkable clinical outcomes in multiple malignant diseases including lung cancer. However, as the dynamic interactions between the cancer cells and the tumor-associated microenvironment evolves, acquired resistance arises. Thus exploration on alternative strategies would be necessary in order to reach long-term tumor suppression. Methods: Murine lung cancer model was established by inoculation of Lewis lung carcinoma cells subcutaneously on the flank of C57BL/6J mice. Chemotherapy (cisplatin or pemetrexed) or anti-PD-1 alone, or in combination started at 7 days post-inoculation as tumor growth was established. Digital caliper was used to measure tumor sizes and survival rates were recorded. Mice were sacrificed upon humane endpoint (tumor size > 17 mm in diameter). Splenocytes and tumor cells were harvested and expression of immune regulatory markers (including PD-L1, PD-1, Tim-3, OX-40, GITR, LAG-3 and CTLA-4) was analyzed by flow cytometry. Results: While PD-1 blockade delayed tumor growth (p< 0.001), there was no significant difference in overall survival. Systemically, anti-PD-1 immunotherapy induced upregulation of Tim-3, OX-40 and GITR; cisplatin alone and in combination with anti-PD-1 induced upregulation of GITR; OX-40 is upregulated in combinations of chemotherapy and anti-PD-1. Population of tumor-infiltrating T cells were increased in anti-PD-1-treated mice, while combination of pemetrexed and anti-PD-1 prompted augmented expression of co-stimulatory receptors including GITR and OX-40 within tumor. Conclusions: Cisplatin and pemetrexed triggered different systematic and intratumoral immune responses when used alone or in combination with anti-PD-1 treatment. Increased expression of co-stimulatory receptors in the tumor in response to combination therapy provide insights for investigation of synergism on anti-PD-1 and alternative therapeutic targets.