Knockout of high-mobility group box 1 in B16F10 melanoma cells induced host immunity-mediated suppression of in vivo tumor growth

Kayoko Waki,Akira Yamada,Hirohisa Yano,Miyako Ozawa,Kanako Yokomizo,Keiko Yamamoto,Sachiko Ogasawara

doi:10.1007/s12032-022-01659-2

Abstract

High-mobility group box 1 (HMGB1) has been reported as a damage-associated molecular pattern (DAMP) molecule that is released from damaged or dead cells and induces inflammation and subsequent innate immunity. However, the role of HMGB1 in the anti-tumor immunity is unclear since inflammation in the tumor microenvironment also contributes to tumor promotion and progression. In the present study, we established HMGB1-knockout clones from B16F10 and CT26 murine tumors by genome editing using the CRISPR/Cas9 system and investigated the role of HMGB1 in anti-tumor immunity. We found that (1) knockout of HMGB1 in the tumor cells suppressed in vivo, but not in vitro, tumor growth, (2) the suppression of the in vivo tumor growth was mediated by CD8 T cells, and (3) infiltration of CD8 T cells, macrophages and dendritic cells into the tumor tissues was accelerated in HMGB1-knockout tumors. These results demonstrated that knockout of HMGB1 in tumor cells converted tumors from poor infiltration of immune cells called “cold” to “immune-inflamed” or “hot” and inhibited in vivo tumor growth mediated by cytotoxic T lymphocytes. Infiltration of immune cells to the tumor microenvironment is an important step in the series known as the cancer immunity cycle. Thus, manipulation of tumor-derived HMGB1 might be applicable to improve the clinical outcomes of cancer immunotherapies, including immune checkpoint blockades and cancer vaccine therapies.

Highlights

High mobility group box 1 (HMGB1) is one of the major chromatin-associated non-histone proteins in the nucleus and acts as a DNA chaperone [1]
B16F10 cells were transfected with pCas-Guide clustered regularly interspaced short palindromic repeats (CRISPR) vector containing HMGB1 guide RNA (gRNA) and linear donor EF1aGFP-P2A-Puro for the genome editing
The in vitro cell proliferation of the HMGB1-knockout clones (A10, G9, G10) was further compared with that of the wild type (WT) B16F10 cells; there were no significant differences between each clone and the WT cells (Fig. 1b)

Summary

Introduction

High mobility group box 1 (HMGB1) is one of the major chromatin-associated non-histone proteins in the nucleus and acts as a DNA chaperone [1]. In addition to its roles in the nucleus and the cytosol, HMGB1 has been reported to be a damage-associated molecular pattern (DAMP) molecule that is released from damaged or dead cells and induces inflammation and subsequent innate immunity [1]. The role of HMGB1 in anti-tumor immunity is complicated, and it is unclear whether HMGB1 has a favorable or unfavorable impact on the host defense against tumors [4]. To clarify this issue, we established HMGB1-knockout clones from B16F10 and CT26 tumor cells by genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, and used these clones to investigate the role of HMGB1 on the anti-tumor immunity

Methods

Results

Conclusion