Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

• Main Authors: Miok Kim, Yong Ki Min, Jinho Jang, Hyejin Park, Semin Lee, Chang Hoon Lee
• Format: Article
• Language: English
• Published: BMJ Publishing Group 2021-07-01
• Series: Journal for ImmunoTherapy of Cancer
• Online Access: https://jitc.bmj.com/content/9/7/e002503.full

Background Although cancer immunotherapy is one of the most effective advanced-stage cancer therapies, no clinically approved cancer immunotherapies currently exist for colorectal cancer (CRC). Recently, programmed cell death protein 1 (PD-1) blockade has exhibited clinical benefits according to ongoing clinical trials. However, ongoing clinical trials for cancer immunotherapies are focused on PD-1 signaling inhibitors such as pembrolizumab, nivolumab, and atezolizumab. In this study, we focused on revealing the distinct response mechanism for the potent CD73 ectoenzyme selective inhibitor AB680 as a promising drug candidate that functions by blocking tumorigenic ATP/adenosine signaling in comparison to current therapeutics that block PD-1 to assess the value of this drug as a novel immunotherapy for CRC.Methods To understand the distinct mechanism of AB680 in comparison to that of a neutralizing antibody against murine PD-1 used as a PD-1 blocker, we performed single-cell RNA sequencing of CD45+ tumor-infiltrating lymphocytes from untreated controls (n=3) and from AB680-treated (n=3) and PD-1-blockade-treated murine CRC in vivo models. We also used flow cytometry, Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS) models, and in vitro functional assays to validate our new findings.Results We initially observed that the expressions of Nt5e (a gene for CD73) and Entpd1 (a gene for CD39) affect T cell receptor (TCR) diversity and transcriptional profiles of T cells, thus suggesting their critical roles in T cell exhaustion within tumor. Importantly, PD-1 blockade significantly increased the TCR diversity of Entpd1-negative T cells and Pdcd1-positive T cells. Additionally, we determined that AB680 improved the anticancer functions of immunosuppressed cells such as Treg and exhausted T cells, while the PD-1 blocker quantitatively reduced Malat1high Treg and M2 macrophages. We also verified that PD-1 blockade induced Treg depletion in AOM/DSS CRC in vivo models, and we confirmed that AB680 treatment caused increased activation of CD8+ T cells using an in vitro T cell assay.Conclusions The intratumoral immunomodulation of CD73 inhibition is distinct from PD-1 inhibition and exhibits potential as a novel anticancer immunotherapy for CRC, possibly through a synergistic effect when combined with PD-1 blocker treatments. This study may contribute to the ongoing development of anticancer immunotherapies targeting refractory CRC.