Materials and Reagents
Tucidinostat (Cat# HY-109015), vorinostat (Cat# HY-10221), TMP-195 (Cat# HY-18361), and NF-κB inhibitor BAY11-7082 (Cat# HY-13453) were obtained from MCE (Monmouth Junction, NJ, USA). Dimethyl sulfoxide (DMSO) have been used as vehicle control for each drug.
4T1 breast cancer cells, Lewis lung cancer (LLC) cells, CT26 colorectal cancer cells, and Raw 264.7 cells were purchased from Chinese Academy of Sciences (Beijing, China) and cultured in RPMI-1640 medium (Hyclone) containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator.
Establishment of murine syngeneic tumor models
For subcutaneous injections, 5 × 105 mouse tumor cells were injected into the right flank of each mouse. When established tumors were palpable 7 days after tumor cell inoculation, mice were treated with different doses of tucidinostat (MCE, gavage, 12.5, 25, 75 mg/kg, oral gavage, daily) and aPD-L1 (BioXcell, Cat# BE0101; 200 μg, intraperitoneal injection, every 3 days). Depletion of CD8+ T cells was performed by intraperitoneal injection of anti-mouse CD8a (BioXcell, Cat# BP0117; 200 μg, every 3 days). Depletion of macrophage was performed by intraperitoneal injection of Clodronate liposomes (FormuMax, Cat# F70101C-A; 1.4 mg/20g body weight, every 3 days), respectively. The volume of tumor nodules was measured every 3 days and calculated as V = (a × b2)/2, where “a” and “b” are the long and short axis of the tumor nodule, respectively. Tumors volume were measured with calipers every three days. Mice were monitored until their individual tumor volume reaches the approved protocol volume limit (2000mm3). At the treatment, the tumor-bearing mice were anesthetized and tissues were harvested for further analysis.
In the previous human clinical trial, 30 mg tucidinostat was given orally twice a week for 4 consecutive weeks in a 4-week cycle. After translating the drug dosage from human to mice, the clinical dose of tucidinostat in mice was near 75mg/kg daily.
Animal studies were conducted in accordance with the NIH animal use guidelines and approved by the Institutional Review Board of National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (Permit Number, NCC2020A167).
Quantitative real-time PCR (RT-qPCR)
Total RNAs were extracted using the RNeasy Kit (Takara Bio). The qRT-PCR was carried out using SYBR Green Premix Ex TaqTM II (Takara Bio) on a ABI StepOnePlus Real-Time PCR Detection System (Thermo Fisher Scientific). Results were normalized to the housekeeping gene GAPDH. Relative gene expression level from different groups were calculated with the 2-ΔΔCT method and compared with the expression level of appropriate control cells.
Speciﬁc primer sequences for individual genes were as follows: CCL5(forward:5′-GTATTTCTACACCAGCAGCAAG-3′;reverse:5′-TCTTGAACCCACTTCTTCTCTG-3′);CXCL9(forward:5′-AATCCCTCAAAGACCTCAAACA-3′;reverse:5′-TCCCATTCTTTCATCAGCTTCT-3′);CXCL10(forward:5′-CAACTGCATCCATATCGATGAC-3′;reverse:5′-GATTCCGGATTCAGACATCTCT-3′);PD-L1(forward:5′-TGAGCAAGTGATTCAGTTTGTG-3′;reverse:5′-CATTTCCCTTCAAAAGCTGGTC-3′);iNOS(forward:5′-GCCGAGTGCAAGCATGGAGAG-3′;reverse:5′-GGCTGTGAGGTGAGGTTGAAGAAG-3′);CD86(forward:5′-ACGGAGTCAATGAAGATTTCCT-3′;reverse:5′-GATTCGGCTTCTTGTGACATAC-3′);CD206(forward:5′-CCTATGAAAATTGGGCTTACGG-3′;reverse:5′-CTGACAAATCCAGTTGTTGAGG-3′);Arg1(forward:5′-CATATCTGCCAAAGACATCGTG-3′;reverse:5′-GACATCAAAGCTCAGGTGAATC-3′);GAPDH(forward:5′-GTATTTCTACACCAGCAGCAAG-3′;reverse:5′-TCTTGAACCCACTTCTTCTCTG-3′).
Western blot analysis
The protein was separated by 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore). Subsequently, the membrane was blocked and incubated overnight at 4°C with the primary antibody including anti-CCL5 mAb (1: 500, Abcam, Cat# ab7198) and anti-phospho-NF-κB p65(1:1000, Cell Signaling Technology, Cat# 3039). The same membrane was probed for GAPDH (1:10000, Abcam, Cat# ab8245) as internal control. Finally, the blots were exposed to the ImageQuant LAS 500 system (GE Healthcare).
Flow cytometric analysis
Tumors from subcutaneous tumor model were harvested for single-cell suspensions using tumor dissociation kit (Miltenyi Biotec GmbH). The drainage lymph node (dLN)s were harvested through mechanical dissociation. Dissociated cells were ﬁltered through a 4 µm strainer and suspended in phosphate-buffered saline (PBS) supplemented with 1% FBS. The cells were stained with the following Abs according to the manufacturer’s instructions: CD45 (Cat# 103114), CD3 (Cat# 100204), CD4 (Cat# 100414), CD8a (Cat# 100752), CD25 (Cat# 101923), CD44 (Cat# 103031), CD62L (Cat# 161203), F4/80 (Cat# 123128), CD11b (Cat# 101245), MHC-II (Cat# 107606), CD206 (Cat# 141708), Gr-1 (Cat# 108423), CD11c (Cat# 117329), CD86 (Cat# 105014), PD-1 (Cat# 135206),or PD-L1 (Cat# 124312) (2.5ul, all from BioLegend) were diluted in FACS buffer (Biolegend).
Various immune cells were separated using a gating strategy based on the expression of known lineage markers for lymphocytes (CD45+), Total T cells (CD45+CD3+), CD4+ T cells (CD45+CD3+CD4+), CD8+ T cells (CD45+CD3+CD8+), Treg cells (CD45+CD3+CD4+CD25+), Central Memory T cells (CD3+CD4+CD44+CD62L+), Effective Memory T cells (CD3+CD4+CD44+CD62L-), Macrophages (CD45+CD11b+F4/80+), M1 macrophages (CD45+CD11b+F4/80+/MHC-II+), DC cells (CD45+CD11b-CD11c+), MDSCs (CD45+CD11b+Gr-1+), and NK cells (CD45+CD3-CD49b+).
Data was performed on the Flow cytometers (BD Biosciences) and analyzed using the Flow Jo software (Ashland, OR, USA).
Tumors were collected and fixed in 4% formalin. Sections of paraffin-embedded tissues (4 μm) were deparaffinized. After antigen retrieval, the sections were incubated with the primary against CD3 (Cat# ab16669), CD4 (Cat# ab183685) or CD8 (Cat# ab217344) (1:200, all from Abcam), and then stained with the HRP linked secondary Abs or fluorophore-conjugated secondary Abs. The cell nucleus were counterstained using hematoxylin or DAPI.
RNA sequencing (RNA-seq) was performed in tumors treated with tucidinostat or controls. Published mRNA signatures for T cells and other cell clusters were analyzed[35, 36]. Hierarchical clustering and Sig Clust were used to identify metagenes of statistical significance and data interpretation.
Mice peripheral blood preparation and Cytokine assay
Peripheral blood were collected from the inner canthus of the experimental mice. Mice white blood cell (WBC) count, red blood cell (RBC) count, platelet (PLT) count, and lymphocytes count were detected by fully automatic hematology analyzer (BC-2800 Vet, Mindray).
Peripheral blood were collected and then centrifuged at 3,000 rpm for 10 min to isolate the serum. The serum alanine transaminase (ALT), alanine transaminase (ALT), and urea nitrogen (BUN) (all from Anoric Bio-technology) were measured using ELISA kits. The serum IL-10 (Cat# 431417), IFN-γ (Cat# 430807), TNF-α (Cat# 430907) (all from Biolegend) and CCL5 (R&D, Cat# DY478) were measured using ELISA kits.
ex vivo chemotaxis assays
Naïve CD8+ T cells were purified from mouse spleen and activated with Dynabeads containing mouse T-activator CD3 (Biolegend, Cat# 100301)/CD28 (Biolegend, Cat# 102101) and recombinant mouse IL-2 (Biolegend, Cat# 714604). And then, the activated CD8+ T cells were seeded in the upper chambers of transwell plates (BD Biosciences) and allowed to migrate for 24 h towards the lower chamber containing medium with different concentrations of CCL5 protein (Pepro Tech, Cat# 250-07).
Bone marrow cells were isolated from femurs and tibias of C57/BL6 mice. 5 × 106 cells per well in 24-well plates were cultured in RPMI-1640 medium containing 10% heated-inactivated fetal bovine serum at 37°C in a 5% CO2 incubator. Bone marrow derived macrophage (BMDM)s were differentiated in the presence of recombinant cytokine M-CSF (20ng/ml, Pepro Tech, Cat# 315-02). Every 2 days, 50% of the medium were replaced with fresh culture medium. After 10 days, we harvested adherent cells and used them for BMDM experiments.
The peripheral blood mononuclear cell (PBMC)s of NSCLC patients were isolated and rested in RPMI-1640 medium containing 10% heated-inactivated fetal bovine serum at 37°C in a 5% CO2 incubator for 6 h. Then, 5 × 105 cells per well in 24-well plates were cultured different doses of tucidinostat as indicated for 24 h.
The cells were stained with the following Abs according to the manufacturer’s instructions: CD14 (Cat# 325604), CD11b (Cat# 393112), CD3 (Cat# 344804), CD4 (Cat# 317428), CD8a (Cat# 344722), CD69 (Cat# 310906), CD86 (Cat# 374208), or HLA-DR (Cat# 307630) (2.5ul, all from BioLegend) were diluted in FACS buffer (Biolegend). Data was collected and analyzed with the Flow cytometers (BD Biosciences) and the Flow Jo software (Ashland, OR, USA).
Various immune cells were separated using a gating strategy based on the expression of known lineage markers for total peripheral blood monocytes (CD14+CD11b+), active peripheral blood monocytes (CD14+CD11b+HLA-DR+/CD14+CD11b+CD86+), total T cells (CD3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), active CD4+ T cells (CD3+CD4+CD69+), and active CD8+ T cells (CD3+CD8+CD69+).
The study was approved by the Institutional Review Board of National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (Permit Number, NCC2020C072).
All data analysis was performed using GraphPad Prism software (version 5.0, GraphPad Software, Inc.). For the comparison among treatment groups in the in vitro and in vivo study, one-way ANOVA was performed. Survival time was defined from the day of tumor cell inoculation until the mice expired naturally or euthanized. Survival curves were drawn using Kaplan-Meier method and compared with log-rank test. P<0.05 was considered statistically significant. In the Figures, symbols were used as: *P<0.05, **P<0.01, ***P<0.001.