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Prevention of liver metastases through perioperative acute CpG-C immune stimulation

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Abstract

Following excision of colorectal tumors, metastatic disease is prevalent, primarily occurs in the liver, and is highly predictive of poor prognosis. The perioperative period is now recognized as critical in determining the incidence of postoperative metastases and long-term cancer outcomes. Thus, various perioperative prophylactic interventions are currently studied during this time frame. However, immune stimulation during the perioperative period has rarely been attempted due to specific contraindications to surgery and various adverse effects. Here, to prevent liver metastases, we perioperatively employed a TLR-9 agonist, CpG-C, which exhibits minimal pyrogenic and other adverse effects in patients. We found that marginating-hepatic (MH) cells in BALB/c mice contained high percentage of NK cells, but exhibited negligible NK cytotoxicity, as previously reported in humans. However, a single CpG-C administration (25-100 µg/mouse) doubled MH-NK cell numbers, increased NK cell activation and maturation markers (NKp46, CD11b), decreased the inhibitory NKG2A ligand, and dramatically increased MH-NK-cell cytotoxicity against the syngeneic CT26 colon cancer line. Moreover, in operated mice, this innocuous intervention also markedly improved resistance to CT26 and MC38 hepatic metastases in BALB/c and C57BL/6 mice, respectively. Beneficial effects of CpG-C were mediated through activation of MH-NK cells, as indicated by an in vivo NK depletion study. Last, CpG-C protected against surgery-induced suppression of MH-NK cytotoxicity and improved their activation indices. Thus, we suggest that systemic perioperative CpG-C treatment should be considered and studied as a novel therapeutic approach to improve long-term cancer outcomes in colorectal cancer patients.

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Abbreviations

CCR7:

Chemokine receptor 7

CpG-C ODN:

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG ODN)

CRC:

Colorectal cancer/carcinoma

CT26:

Colon tumor cell line

IFN:

Interferon

MC38:

Mouse colon cell line

MH-NK cell:

Marginating-hepatic NK cell

MP-NK cell:

Marginating pulmonary NK cell

MRD:

Minimal residual disease

NKCC:

NK cells cytotoxicity

pDC:

plasmacytoid dendritic cells

TLR9:

Toll-like receptor 9

TRAIL:

TNF-related apoptosis-inducing ligand

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Funding

This work was supported by NIH/NCI Grant # CA125456 and CA172138 (SBE) and by the Israel-USA bi-national Science Foundation # 2005331 (SBE & GGP).

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Authors and Affiliations

  1. Neuroimmunology Research Unit, Sagol School of Neuroscience, The School of Psychological Sciences, Tel Aviv University, 69978, Tel Aviv, Israel

    Liat Sorski, Rivka Melamed, Ben Levi, Pini Matzner, Hagar Lavon, Ella Rosenne, Lee Shaashua, Itay Ricon, Elad Sandbank, Amit Benbenishty & Shamgar Ben-Eliyahu

Authors
  1. Liat Sorski
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  2. Rivka Melamed
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  3. Ben Levi
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  4. Pini Matzner
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  5. Hagar Lavon
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  6. Ella Rosenne
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  7. Lee Shaashua
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  8. Itay Ricon
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  10. Amit Benbenishty
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  11. Shamgar Ben-Eliyahu
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Contributions

LS is the lead researcher of the studies presented in this paper, and is reponsible for designing, initiating and performing all the experiments presented herein, analyzing their data, and writing the paper itslef. RM assisted performing both the in vivo and ex vivo experiments and helped analysing their data. BL assisted performing the in vivo experiments. PM assisted performing the in vivo experimnts, and analyzing the ex vivo expreiments data. HL assisted performing the in vivo experiments. ER assisted in cells lines preperation and handling, and in analyzing the data. LS assisted performing the in vivo experiments. IR assisted in analyzing the data. ES assisted in performing the in vivo experiments. AB assisted in analyzing the data. SB-E is the principal investigator of the lab and is involved in the design of the experiments, in performing and analyzing their data, and in advising and assisting in writing the paper.

Corresponding author

Correspondence to Shamgar Ben-Eliyahu.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

The Institutional Animal Care and Use Committee of Tel Aviv University approved all studies.

Approval numbers for the experiments

CT26 – P-13-014, MC38 – 10-18-007.

Animal source

BALB/c male and female mice and C57B1/6j male and female mice were purchased from Harlan laboratories (Jerusalem, Israel) at the age of 4 weeks. Animals were housed 3–4 per cage at 22 ± 1 °C, on a 12:12 light/dark cycle and were allowed ad libitum access to food and water. Animals were used at the age of 8–12 weeks. (Animals were age-matched between groups within each experiment.)

Cell line authentication

The CT26 tumor cell line: CT26 tumor cells were kindly provided by Prof. Eliezer Flesher (Department of Human Microbiology, Faculty of Medicine, Tel Aviv University).

The MC38 tumor cell line

The MC38 murine colon adenocarcinoma was derived from tumor that arose in C57B1/6 mice. Tumor cells were kindly provided by Dr. Eran Nizri (Tel Aviv Sourasky Medical center).

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Sorski, L., Melamed, R., Levi, B. et al. Prevention of liver metastases through perioperative acute CpG-C immune stimulation. Cancer Immunol Immunother 69, 2021–2031 (2020). https://doi.org/10.1007/s00262-020-02596-7

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  • DOI: https://doi.org/10.1007/s00262-020-02596-7

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