Combination Therapy of Mithramycin A and Immune Checkpoint Inhibitor for the Treatment of Colorectal Cancer in an Orthotopic Murine Model.

Rinku Dutta,Srinivas Bharadwaj,Ryan Green,Subhra Mohapatra,Mark Howell,Shyam S Mohapatra,Roukiah Khalil,Karthick Mayilsamy

doi:10.3389/fimmu.2021.706133

Abstract

The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and blocking this axis has been found to be effective in a subset of patients. Although blocking PD-L1 has been shown to be effective in 5–10% of patients, the majority of the cohorts show resistance to this checkpoint blockade (CB) therapy. Multiple factors assist in the growth of resistance to CB, among which T cell exhaustion and immunosuppressive effects of immune cells in the tumor microenvironment (TME) play a critical role along with other tumor intrinsic factors. We have previously shown the polyketide antibiotic, Mithramycin-A (Mit-A), an effective agent in killing cancer stem cells (CSCs) in vitro and in vivo in a subcutaneous murine model. Since TME plays a pivotal role in CB therapy, we tested the immunomodulatory efficacy of Mit-A with anti-PD-L1 mAb (αPD-L1) combination therapy in an immunocompetent MC38 syngeneic orthotopic CRC mouse model. Tumors and spleens were analyzed by flow cytometry for the distinct immune cell populations affected by the treatment, in addition to RT-PCR for tumor samples. We demonstrated the combination treatment decreases tumor growth, thus increasing the effectiveness of the CB. Mit-A in the presence of αPD-L1 significantly increased CD8+ T cell infiltration and decreased immunosuppressive granulocytic myeloid-derived suppressor cells and anti-inflammatory macrophages in the TME. Our results revealed Mit-A in combination with αPD-L1 has the potential for augmented CB therapy by turning an immunologically “cold” into “hot” TME in CRC.

Highlights

Colorectal cancer (CRC) is the third most common cancer in men, the second most common in women, and the second most common cause of cancer-related deaths in the USA [1]
We have previously reported Mit-A acts as an inhibitor of CRC by targeting the cancer stem cells (CSCs) [23]
We aimed to determine whether MitA along with anti-PD-L1 mAb (aPD-L1) can cause increased cell apoptosis using orthotopic tumor biopsies grown as monolayer culture and compared with MC38 cell line

Summary

Introduction

Colorectal cancer (CRC) is the third most common cancer in men, the second most common in women, and the second most common cause of cancer-related deaths in the USA [1]. With an estimated 5% lifetime risk, CRC is one of the malignant cancers whose 5-year survival rate is poor when patients are diagnosed at a late stage [2, 3]. Microsatellite instability (MSI) plays a pivotal role in CRC stages and arises due to deficiencies in the DNA mismatch repair system, causing insertion, deletion, or misincorporation of nucleotides in the DNA [4]. Recent advances in checkpoint blockade (CB) therapy for microsatellite instability (MSI) positive CRC patients have shown dramatic response for patients with high MSI (MSI-H) [5]. Single-agent checkpoint inhibitors do not show response in CRC patients with microsatellite stable (MSS) carcinomas, which comprise the majority of the aggressive CRCs with poor outcomes [5, 8]

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