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

Abstract

Background Cetuximab 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. Methods Ex 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. Results We 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. Conclusions This 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.

NK cell-derived exosomes enhance the anti-tumor effects against ovarian cancer by delivering cisplatin and reactivating NK cell functions

“…We previously established a method to obtain eNK cells from PBMC by co-culturing with K562-mBIL-21 feeder layer cells (Lifeark, China) for 2 weeks ( 23 ). Compared with other allogeneic cell sources, such as bone marrow and peripheral blood, cord blood (CB) is readily available as a frozen, “off the shelf” product, which provides more advantages as the starting material for cell therapies.…”

“…The cord blood mononuclear cells (CBMC) were isolated from the cord blood of 3 healthy donors by density gradient centrifugation. eNK were obtained by co-culturing CBMC with irradiation-inactivated K562 engineered cells for 14 days in the presence of IL-2 ( 23 ). The human ascites mononuclear cells were isolated from the ascites of 2 patients with malignant OC by Ficoll gradient density centrifugation, and the NK cells in ascites (AS_NK) were obtained using EasySep™ Human NK Cell Isolation Kit (Stemcell Technologies).…”

Emerging role of RNA sensors in tumor microenvironment and immunotherapy

“…Another clinical trial of pancreatic cancer demonstrated that the combination of peptide pulsed DCs and poly-ICLC was safe and could induce a measurable tumor specific T cell population [ 150 ]. Additionally, reovirus-activated NK cells combined with cetuximab could synergistically enhance their antitumor cytotoxicity in colorectal cancer, which was dependent on TLR3 and its downstream signals [ 151 ].…”

NK Cell-Based Immunotherapy in Colorectal Cancer

“…Along this line, a recent study by Long and colleagues demonstrated that NK cells exposed to Cetuximab in combination with oncolytic reovirus were activated mainly by TLR-3 and showed increased ADCC against CRC lines in vitro independently to KRAS mutation status and EGFR expression level. Importantly, the combined exposure to reovirus and Cetuximab provided a greater NK-mediated anti-tumor effect than monotherapy with Cetuximab in vivo [ 182 ].…”