Reovirus enhances cytotoxicity of natural killer cells against colorectal cancer via TLR3 pathway

Shiqi Long,Weiwei Ouyang,Nianxue Wang,Zhixu He,Xiaojin Lin,Chunxiang Liao,Xiaoqing Chen,Yuanyuan An,Xianyao Wang,Xing Zhao,Yangzhuo Gu

doi:10.1186/s12967-021-02853-y

Abstract

BackgroundCetuximab has been approved for use for first-line treatment of patients with wild-type KRAS metastatic colorectal cancer (CRC). However, treatment with cetuximab has shown limited efficacy as a CRC monotherapy. In addition, natural killer (NK) cell function is known to be severely attenuated in cancer patients. The goal of this study was to develop a new strategy to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells, in combination with cetuximab against CRC cells.MethodsEx vivo expanded NK cells were stimulated with reovirus, and reovirus-activated NK cells mediated ADCC assay were performed on CRC cells in combination with cetuximab. The synergistic antitumor effects of reovirus-activated NK cells and cetuximab were tested on DLD-1 tumor-bearing mice. Finally, Toll-like receptor 3 (TLR3) knockdown in NK cells, along with chemical blockade of TLR3/dsRNA complex, and inhibition of the TLR3 downstream signaling pathway, were performed to explore the mechanisms by which reovirus enhances NK cell cytotoxicity.ResultsWe first confirmed that exposure of NK cells to reovirus enhanced their cytotoxicity in a dose-dependent manner.We then investigated whether reovirus-activated NK cells exposed to cetuximab-bound CRC cells exhibited greater anti-tumor efficacy than either monotherapy. Co-culture of CRC cell lines with reovirus-activated NK cells indicated that NK cytotoxicity was significantly higher in combination with cetuximab, regardless of KRAS mutation status or EGFR expression level. We also found that reovirus activation of NK cells, in conjunction with cetuximab, resulted in significantly stronger anti-tumor efficacy.Finally, TLR3 knockdown, inhibition of TLR3/dsRNA complex or TBK1/IKKε demonstrated that activation of NK cells by reovirus was dependent on TLR3 and its downstream signaling pathway.ConclusionsThis study demonstrated that combination treatment of reovirus-activated NK cells with cetuximab synergistically enhances their anti-tumor cytotoxicity, suggesting a strong candidate strategy for clinical treatment of CRC.

Highlights

Cetuximab has been approved for use for first-line treatment of patients with wild-type KRAS metastatic colorectal cancer (CRC)
Using multiple CRC cell lines with different KRAS mutation status and epidermal growth factor receptor (EGFR) expression levels we found that reovirus directly enhanced natural killer (NK) cell cytotoxicity,and that, in combination with cetuximab, reovirus-activated NK cells exhibited increased CRC cell killing in vitro,regardless of KRAS mutation or EGFR expression. using human colorectal tumor xenograft models, we observed that reovirus activation of NK cells, in conjunction with cetuximab, provided significantly greater anti-tumor effects than either monotherapy
As CD69 is a marker for NK cell activation, we assessed their activation through flow cytometry assays, which confirmed that CD69 was up-regulated in response to reovirus stimulation (Fig. 1b, c).Perforin (PRF1) and granzymes represent two distinct categories of cytotoxic proteins released by NK cells

Summary

Introduction

Cetuximab has been approved for use for first-line treatment of patients with wild-type KRAS metastatic colorectal cancer (CRC). For NK cells, recognition of IgG1 mAbs by FcγRIIIa (CD16) results in enhanced NK cell degranulation, cytokine secretion, and cytotoxicity [8, 9].despite cetuximab’s multiple mechanisms of action, responses to cetuximab are limited: only 1 in 5 patients with metastatic CRC responds to cetuximab [10].New approaches are needed to enhance its efficacy. To this end, increasing the NK cell response to cetuximab therapy could potentially enhance the effects of ADCC in CRC therapy

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