Altered expression of immune regulatory receptors upon combination of chemotherapy and checkpoint blockade immunotherapy in a lung cancer mouse model

Abstract

Introduction: Lung cancer remains as the most lethal cancer disease in Hong Kong. Although blockade of inhibitory immune checkpoints achieved remarkable clinical outcomes, acquired immune resistance arises as dynamic interactions between the cancer cells and the tumour-associated microenvironment evolve. Exploration on alternative strategies to battle against lung cancer is therefore necessary for long-term tumour 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 antibody to programmed cell death protein 1 (anti-PD-1) alone, or in combination started at 7 days post-inoculation for 2 weeks. Tumour growth was measured by digital calliper and survival rate was recorded. Mice were sacrificed when they reached the humane endpoint. Splenocytes and tumour 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 analysed by flow cytometry. Results: While PD-1 blockade delayed tumour growth (P<0.001), no significant difference in overall survival was observed. Systemically, anti-PD-1 immunotherapy induced upregulation of Tim-3, OX-40 and GITR; cisplatin ± anti-PD-1 induced upregulation of GITR; whilst OX-40 is induced in both combinations of chemotherapy and anti-PD-1. Tumour-infiltrating T cells were increased in anti-PD-1–treated mice, while combination of pemetrexed and anti-PD-1 induced upregulation of co-stimulatory receptors including GITR and OX-40 within tumour. Conclusion: Cisplatin and pemetrexed induced different systematic and intratumoural immune responses when used alone or in combination with anti-PD-1 treatment. Increased expression of co-stimulatory receptors in the tumour in response to combination therapy provide insights for investigation of synergism on anti-PD-1 and alternative therapeutic targets. Acknowledgement This study was supported by YC Chan Scientist Award 2019 and Donation from Lifestyle International Holdings Limited.