Knockout of High-Mobility Group Box 1 in B16F10 Melanoma Cells Induced Host Immunity-Mediated Suppression of In Vivo Tumor Growth

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.

Suppression of high mobility group box 1 in B16F10 tumor does not inhibit the induction of neoantigen‐specific T cells

“…We next investigated whether neoantigen‐reactive T cells were present in B16F10 tumor‐bearing mice. We previously established HMGB1‐KO clones of B16F10 cells, and the in vivo tumor growth of these clones was reported to be markedly suppressed by CD8‐mediated host immunity; 21 thus, the WT as well as the HMGB1‐KO clones (G9 and A10E2) of B16F10 cells were used in the present experiment. Spleen cells were obtained from tumor‐bearing mice or control non‐tumor bearers on day 9; after 6 days of in vitro culture with one of the neoantigen peptides, the cells were subjected to IFN‐γ ELISPOT assay (Figure 2).…”

“…The cells were cultured in high glucose D‐MEM (Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% FCS (Thermo Fisher, Waltham, MA), L‐glutamine, and 50 μg/ml gentamicin at 37°C in a 5% CO 2 incubator. The B16F10‐derived HMGB1‐KO clones (G9 and A10E2) were previously established in our laboratory 21 . The linear donor was inserted around cDNA position 198 in exon 2 of the HMGB1 gene, which was 25 nucleotides downstream of the initiation codon, and all the functional domains of HMGB1 were disrupted in these KO clones.…”

“…Our previous study 22 as well as others 23–25 regarding the anti‐tumor effect of HMGB1 inhibitors, suggested a possible application of suppression of tumor‐derived HMGB1 to cancer immunotherapy, such as ICB and neoantigen vaccine therapies 21–25 . Although most previous studies supported the negative effect of HMGB1 on anti‐tumor immunity, 21–25 a positive role of HMGB1 in the initiation of innate and subsequent adaptive immunity has also been shown 26,27 . Namely, the binding of HMGB1 to receptors, such as Toll‐like receptor (TLR)‐2, TLR‐4, and the receptor for advanced glycation end products (RAGE), on macrophages and dendritic cells induces proinflammatory cytokines through activation of interferon regulatory factor‐3 (IRF3) and subsequently triggers innate immunity 27,28 .…”

“…The effects of tumor‐derived DAMPs on anti‐tumor immunity have not been well understood. We recently reported that high mobility group box 1 (HMGB1), a representative DAMP molecule, showed a negative impact on anti‐tumor immunity 21 . Namely, knockout (KO) of HMGB1 in the tumor cells using CRIPR/Cas9 suppressed in vivo tumor growth mediated by host CD8 T cells and accelerated infiltration of T cells as well as macrophages and dendritic cells (DCs) into the tumor tissues 21 .…”

“…We recently reported that high mobility group box 1 (HMGB1), a representative DAMP molecule, showed a negative impact on anti‐tumor immunity 21 . Namely, knockout (KO) of HMGB1 in the tumor cells using CRIPR/Cas9 suppressed in vivo tumor growth mediated by host CD8 T cells and accelerated infiltration of T cells as well as macrophages and dendritic cells (DCs) into the tumor tissues 21 . Our previous study 22 as well as others 23–25 regarding the anti‐tumor effect of HMGB1 inhibitors, suggested a possible application of suppression of tumor‐derived HMGB1 to cancer immunotherapy, such as ICB and neoantigen vaccine therapies 21–25 .…”