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

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 Malat1^high 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.