Nano-Regulator Inhibits Tumor Immune Escape via the "Two-Way Regulation" Epigenetic Therapy Strategy

Abstract

Tumor immune escape caused by low levels of tumor immunogenicity and immune checkpoint-dependent suppression limits the immunotherapeutic effect. Herein, a "two-way regulation" epigenetic therapeutic strategy is proposed using a novel nano-regulator that inhibits tumor immune escape by upregulating expression of tumor-associated antigens (TAAs) to improve immunogenicity and downregulating programmed cell death 1 ligand 1 (PD-L1) expression to block programmed death-1 (PD-1)/PD-L1. To engineer the nano-regulator, the DNA methyltransferase (DNMT) inhibitor zebularine (Zeb) and the bromodomain-containing protein 4 (BRD4) inhibitor JQ1 are co-loaded into the cationic liposomes with condensing the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine (CpG) via electrostatic interactions to obtain G-J/ZL. Then, asparagine-glycine-arginine (NGR) modified material carboxymethyl-chitosan (CMCS) is coated on the surface of G-J/ZL to construct CG-J/ZL. CG-J/ZL is shown to target tumor tissue and disassemble under the acidic tumor microenvironment (TME). Zeb upregulated TAAs expression to improve the immunogenicity; JQ1 inhibited PD-L1 expression to block immune checkpoint; CpG promote dendritic cell (DC) maturation and reactivated the ability of tumour-associated macrophages (TAM) to kill tumor cells. Taken together, these results demonstrate that the nano-regulator CG-J/ZL can upregulate TAAs expression to enhance T-cell infiltration and downregulate PD-L1 expression to improve the recognition of tumor cells by T-cells, representing a promising strategy to improve antitumor immune response.

Keywords: epigenetic regulation; nano-regulator; tumor immune escape; two-way regulation.

Scheme 1

Schematic illustration of the integrated nano‐regulator inhibits tumor immune escape via the “two‐way regulation” epigenetic therapy strategy. a) The assembly and disassembly of the nano‐regulator CG‐J/ZL. b) The “two‐way regulation” function of CG‐J/ZL in vivo. Under the mediation of NGR, CG‐J/ZL could target tumor tissue and trigger disassembly under the acidic TME. Zeb can effectively upregulate TAAs to enhance tumor immunogenicity; JQ1 can inhibit PD‐L1 expression to block immune checkpoint blockade; CpG can promote DC maturation that would cooperate with Zeb to promote activation of T‐cells. Besides, CpG could reactivate the ability of TAM to kill tumor cells.

Figure 1

Evaluation of the assembly and disassembly behavior of CG‐J/ZL. a) Scheme of assembly and disassembly behavior for CG‐J/ZL. b) Sizes, zeta potentials and morphologies of Blank Lip, G‐J/ZL, CG‐J/ZL. c) Size and d) zeta potential of CG‐J/ZL at different pH. In vitro release behavior of e) JQ1 and f) Zeb. g) The size, PDI and h) zeta potential of CG‐J/ZL during 7 days (n = 3, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001).

Figure 2

CG‐J/ZL enhanced drugs accumulation at tumor tissue and improved the co‐delivery efficiency of drugs. a) In vivo imaging of 4T1‐bearing mice. b) Ex vivo imaging and (c) total fluorescence intensity of main organs and tumors. d) Fluorescence images and e) flow cytometric analysis of cellular uptake. f–h) flow cytometric analysis of co‐delivery efficiency. (n = 3, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001).

Figure 3

CG‐J/ZL achieved “two‐way regulation” via upregulating TAAs expression and downregulating PD‐L1 expression. a) Immunofluorescence staining analysis of BRD4 and DNMT1 in 4T1 cells. Quantitative analysis of b) BRD4 and c) DNMT1 in 4T1 cells. d–f) ELISA analysis of TAAs expression (TRP1, MAGE‐E1, and CD146) in 4T1 cells. g,h) PD‐L1 expression on 4T1 cells. i,j) BMDC maturation. k,l) The ratio of M1‐TAM and M2‐TAM. m) The ratio of M1‐TAM/M2‐TAM. (n = 3 ^# p < 0.05, ^## p < 0.01, ^### p < 0.001).

Figure 4

CG‐J/ZL enhanced antitumor immune response in 4T1‐bearing mice. a–c) The percentage of CD4+ T‐cells and CD8+ T‐cells in 4T1 tumor tissues. d) Relative ratio of M1‐TAM and M2‐TAM. e) The M1‐TAM/M2‐TAM ratio. f,g) DC maturation. h,i) Percentage of CTLs infiltrated in 4T1 tumor tissues. j) The levels of cytokines in tumor tissue. (n = 3, ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001).

Figure 5

The nano‐regulator CG‐J/ZL enhanced the antitumor efficacy in 4T1‐bearing mice. a) Schedule of in vivo anti‐tumor efficacy. b) Tumor volume and c) body weight of 4T1‐bearing mice. d) Tumor weight and e) tumor images of ex vivo tumors. f) Immunohistochemical images of tumor tissue sections. (n = 6, ^** p < 0.01, ^*** p < 0.001, compared with CG‐J/ZL. ^# p < 0.05, compared with P+CG‐J/ZL. ^@ p < 0.05, ^@@ p < 0.01, and ^@@@ p<0.001, compared with C+Z+J. ^&& p < 0.01, ^&&& p < 0.001, compared with CpG+Zeb).

Figure 6

The nano‐regulator CG‐J/ZL exhibited good anti‐metastasis effect. a) The experiment schedule of anti‐metastasis in 4T1‐bearing mice. b) Tumor volume and c) body weight of 4T1 bearing mice. d) Tumor weight and e) tumor photographs. f) Immunohistochemical staining of tumor sections. g) Relative fluorescence intensity of lung. h) Lung weight. i) Ex vivo bioluminescence images of lungs. j) H&E staining images of lungs. (n = 6, ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001).