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PD-L1 Targeted Immunoliposomes with PD-L1 siRNA and HDAC Inhibitor for Anti-Lung Cancer Immunotherapy
Biomed Sci Letters 2022;28:247-259
Published online December 31, 2022;  https://doi.org/10.15616/BSL.2022.28.4.247
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Se-Yun Hong* , Seong-Min Lee* , Pyung-Hwan Kim* * and Keun-Sik Kim†,* *

Department of Biomedical Laboratory Science, Konyang University, Daejeon 35365, Korea
Correspondence to: Keun-Sik Kim. Department of Biomedical Laboratory Science, Konyang University, Daejeon 35365, Korea.
Tel: +82-42-600-8434, Fax: +82-42-600-8408, e-mail: kskim11@konyang.ac.kr
*Graduate student, **Professor.
Received October 14, 2022; Revised November 23, 2022; Accepted November 28, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Immunotherapy, which uses an immune mechanism in the body, has received considerable attention for cancer treatment. Suberoylanilide hydroxamic acid (SAHA), also known as a histone deacetylase inhibitor (HDACi), is used as a cancer treatment to induce active immunity by increasing the expression of T cell-induced chemokines. However, this SAHA treatment has the disadvantage of causing PD-L1 overexpression in tumor cells. In this study, we prevented PD-L1 overexpression by blocking the PD-1/PD-L1 pathway using PD-L1 siRNA. We designed two types of liposomes, the neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin (POPC) for SAHA, and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) for siRNA. To effectively target PD-L1 in cancer cells, we conjugated PD-L1 antibody with liposomes containing SAHA or PD-L1 siRNA. These immunoliposomes were also evaluated for cytotoxicity, gene silencing, and T-cell-induced chemokine expression in human non-small cell lung cancer A549 cells. It was confirmed that the combination of the two immunoliposomes increased the cancer cell suppression efficacy through Jurkat T cell induction more than twice compared to SAHA alone treatment. In conclusion, this combination of immunoliposomes containing a drug and nucleic acid has promising therapeutic potential for non-small-cell lung carcinoma (NSCLC).
Keywords : Immunotherapy, Liposomes, Non-small-cell lung carcinoma, PD-L1, SAHA
INTRODUCTION

There are various therapeutic modalities for cancer treatment, such as surgery, chemotherapy, radiation therapy, targeted therapy, and immunotherapy. While most cancer therapies attack cancer cells directly, immunotherapy increases immune responses and induces the death of cancer cells via the host's immune system. Hence, immunotherapy is a promising cancer treatment method (Lian et al., 2019). However, cancer cells continue to grow owing to immune responses in the tumor microenvironment (TME) and resist immune cells via the immune checkpoint pathway, which inactivates the immune system (Sanmamed and Chen, 2018). The programmed cell death protein 1 pathway (PD-1/PD-L1) in tumors, including non-small cell lung cancer (NSCLC), presents an immune evasion mechanism from adaptive immunity (Wang et al., 2015). This interaction of PD-L1 with PD-1 induces T-cell neutralization by transmitting a negative signal to T-cell activity (Friedman and Postow, 2016). Immune checkpoint inhibitors, such as the blockade of CTLA-4 (cytotoxic T lymphocyte associated Antigen 4) and PD-1 (programmed cell death protein 1) currently used as T-cell mediated anticancer therapy by blocking CTLA-4 and PD-1 expressed in T cells (Zheng et al., 2016; Wei et al., 2018).

Histone deacetylase inhibitors (HDACi) inhibit growth, apoptosis, and cell cycle arrest in tumor cells, including NSCLC cells, wherein suberoylanilide hydroxamic acid (SAHA; vorinostat) could serve as an anticancer agent (Zhao et al., 2014; Woods et al., 2015). SAHA, approved by the FDA in 2006, is a drug used to treat cutaneous T-cell lymphoma (CTCL). Interestingly, HDACi-treated tumor cells are known to express various chemokines, such as CCL5 and CXCL10, which derive active immunity to control T-cell migration into the tumor microenvironment. The expression of chemokines allows for more effective immunotherapy by recruiting T cells into the tumor microenvironment (Hopewell et al., 2013). Despite its therapeutic potential, SAHA is difficult to use in vivo for intravenous administration owing to its low pharmacokinetics, bioavailability, and poor aqueous solubility (Hrzenjak et al., 2010). In addition, HDAC inhibition upregulates PD-L1 expression in tumor cells, which cannot induce active immunotherapy owing to the interaction with T cells. To overcome these limitations, they are used in combination with other drugs.

Currently, siRNA is used to treat a variety of diseases by targeting only specific genes at the mRNA level (Haghiralsadat et al., 2018). However, siRNA delivery is still under development to reduce toxicity and protect cells against degradation. Naked siRNAs are typically difficult to deliver effectively into cells and are easily destroyed in the bloodstream (Chen et al., 2018). For the effective delivery of genes such as siRNAs, several requirements must be met: first, the siRNA must be condensed to a minimum size in the carrier, and then protected from degradation by other enzymes. It also induced selective gene silencing without toxicity (Youngren-Ortiz et al., 2017). Viral vector-mediated methods have been proposed for effective gene delivery; however, they have many drawbacks that can lead to severe immunogenicity (Bolhassani and Rafati, 2011; Wirth and Ylä-Herttuala, 2014). To overcome these shortcomings, various carriers have been developed for siRNAs based on non-viral vectors, such as cationic nanoparticles and liposomes, through the characteristic electrostatic bonding of negative charges (Bhavsar et al., 2016).

In this regard, liposomes, which are widely used as non-viral vectors, have gained increasing importance over the decades owing to their superior ability to induce cell internalization. In addition, they can overcome cell toxicity and drug resistance, which are the shortcomings of free drugs (Nag and Awasthi, 2013). In particular, cationic liposomes are mainly used for gene delivery because they easily form complexes with nucleic acids, leading to excellent cell uptake. However, they are concerned about inducing non-specific targeting in non-target cells in vivo (Audouy et al., 2002). Therefore, targeting ligands, such as peptides, aptamers, and antibodies, are mainly used to conjugate the liposome surface, which is referred to as active targeting. Ligands selectively target tumor cells through receptor-mediated endocytosis mechanisms and can enhance anticancer effects in comparison with free drugs or passive targeting liposomes (Noble et al., 2014). They can also effectively deliver nucleic acids to cells.

In this study, to target A549, a lung cancer cell line, PD-L1 antibody was used and ligated with liposomes containing SAHA or siRNA. As a result, combination therapy with SAHA and siRNA-conjugated antibody-binding liposomes overcame the limitations of anticancer effects with only chemical or nucleic acid treatment. In addition, drug delivery could be further enhanced by targeting with antibodies, and T-cell induced chemokines and tumor cell killing were shown to be effective in PD-L1 expressing NSCLC.

MATERIALS AND METHODS

Reagents

DSPE-PEG2000[1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethylene-glycol)-2000], DSPE-PEG2000-Mal, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoenthanolamine (Rho-DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin (POPC), and cholesterol were purchased from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). Liposome extruder and Polycarbonate membranes (800, 400, 200 and 100 nm) were purchased from Whatman (Kent, Maidstone, UK). Agarose beads (Sepharose TM CL-4B) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 30 k Amicon tube was purchased from Merck Millipore (Billerica, MA, USA). Slide-A-Lyzer Dialysis Cassette were purchased from Thermo Scientific (Waltham, MA, USA). SAHA, PMA (phorbol 12-myristate 13-acetate) and Ionomycin were purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO).

Preparation of micelles conjugated with anti-PD-L1 antibody

To product thiolated antibody, anti-PD-L1 antibody was mixed with Traut's reagent (Thermo) for 1 h at RT at 1:10 molar ratio in HEPES-buffered saline (HBS) (25 mM HEPES, 140 mM NaCl, 2 mM EDTA, pH 8.0). The unreacted Traut's reagent was removed by PD-10 desalting column (GE healthcare) with HBS (25 mM HEPES, 140 mM NaCl, pH 7.5). The thiolated antibody was then mixed with DSPE-PEG2000/DSPE-PEG2000-maleimide (micelle) (1:4 molar ratio) at 1:10 molar ratio and the mixture was incubated overnight at 4℃. The free antibodies and micelles were removed using PD-10 desalting column in HBS (pH 7.5).

Preparation of Immuno-DCD[siRNA] liposomes and Immuno-PCD[SAHA] liposomes

First, to prepare DCD liposome, DOTAP, cholesterol, and DSPE-PEG2000 were dissolved in chloroform and methanol (2:1, v/v). The chloroform-methanol mixture was evaporated by N2 gas to produce a lipid thin film. Incubation was carried out for 1 h using a vacuum pump to remove residual organic solvent. The film was hydrated using pure water. The hydrated solution was sonicated for 3 times and extruded 10 times through Whatman nucleopore polycarbonate membranes using pore size from 800 nm to 100 nm on an Avanti-Extruder. After then, cationic liposomes were mixed with PD-L1 siRNA (4:1 N/P ratio) and incubated for 30 min at room temperature. The cationic lipoplexes containing PD-L1 siRNA were referred to as DCD[siRNA].

Secondly, to prepare PCD liposomes, POPC, cholesterol, and DSPE-PEG2000 were dissolved in chloroform and methanol (2:1, v/v). And then, the mixture was evaporated by N2 gas to produce a lipid thin film. Incubation was carried out for 1 h using a vacuum pump to remove residual organic solvent. SAHA was dissolved in HBS (pH 7.5), this solution was used to hydrate the dried lipids and stirred constantly for 30 min at 60℃. To remove unencapsulated SAHA, dialysis was performed by using Slide-A-Lyzer cassette with 20 K MWCO (Thermo) according to manufacturer protocol. The solution was extruded through polycarbonate membranes with a pore diameter of 200 nm. The PCD liposomes encapsulating SAHA were referred to as PCD[SAHA]. Finally, micelles conjugated with anti-PD-L1 antibody were post-inserted into the prepared all liposomes by incubation for 1 h at 60℃. The anti-PD-L1 antibody-conjugated DCD [siRNA] and PCD[SAHA] were referred to as Immuno-DCD[siRNA] and Immuno-PCD[SAHA].

Cell culture

Human non-small-cell lung cancer (NSCLC) A549 cells and Jurkat T cells (clone E6-1) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco/Invitrogen, Carlsbad, CA, USA), 1% penicillin/streptomycin (Hyclone, Logan, UT, USA) in a humidified atmosphere with 5% CO2 at 37℃.

Cell viability assay (Tumor cells inhibition assay)

A549 cells (2×104 cells/well) were seeded in 96-well plate and incubated 24 h. The cells were then treated with various concentration of SAHA for 24 and 48 h. To evaluate cytotoxicity of PCD[SAHA], liposomes (POPC:cholesterol: DSPE-PEG) encapsulated with SAHA or immuno-PCD [SAHA], PCD[SAHA] conjugated antibody, A549 cells (1 ×104 cells/well in 96-well plate) were incubated with indicated concentration of for 24 and 48 h. After incubation for 24 and 48 h, the medium was exchanged with a fresh medium containing EZ-Cytox reagent 10 μL (Daeil lab service, Seoul, South Korea) and incubated for 2 h at 37℃. The absorbance was measured at 450 nm by using a Versa-Max microplate reader (Molecular Device, Sunnyvale, CA, USA).

Apoptosis assay

To determine the effect of SAHA, A549 cells (8×104 cells/well in 24-well plate) were determined using annexin V apoptosis kit. The cells were treated with the indicated concentration of SAHA for 48 h. After 48 h, the cells were washed twice with PBS and the cells were counted and 1× 105 cells were resuspended in 100 μL of binding buffer. And then, addition of 5 μL annexin V-FITC and 5 μL PI (BD Biosciences, San Jose, CA, USA) and incubation for 15 min at room temperature in the dark. Subsequently, 400 μL of binding buffer were added and the early and late apoptotic cells were analyzed by NovoCyte Flow Cytometer (ACEA Bioscience, San Diego, CA, USA). All tests were performed in triplet.

Enzyme-linked immunosorbent assay

A549 cells (2×105 cells/well in 6-well plates) were treated with the indicated concentration of SAHA for 48 h. The cell lysates and supernatants were harvested and the secretion levels of PD-L1 and CCL5 in cell culture were assayed using a DuoSet ELISA kit (R&D systems, Minneapolis, MN, USA) following the manufacturer's protocols. The absorbance was measured at 450 nm by using a microplate reader. All assays were performed as three independent experiments.

Analysis of PD-L1 silencing by PD-L1 siRNA transfection

The expression rate of PD-L1 was confirmed by ELISA by the transfection of siRNA to prevent the expression of PD-L1 increased by SAHA. A549 cells (2×105 cells/well in 6-well plate) were transfected with predesigned PD-L1 siRNA (No. 29126-1) (Bioneer Inc., Daejeon, South Korea) using a cationic liposome for 4 h in serum free media and incubated for 24 h in fresh media prior to incubation with standard condition or 10 μM SAHA for 48 h. And then, PD-L1 knockdown in A549 cells was confirmed by ELISA.

To evaluate PD-L1 protein silencing effect of immuno-DCD[siRNA], an antibody-conjugated form of PD-L1 siRNA and liposome complex (DOTAP:cholesterol:DSPE-PEG), A549 cells (6×104 cells/well in 24-well plate) were transfected with immuno-DCD encapsulating PD-L1 siRNA in serum-free media for 4 h and incubated for 24 h in fresh media prior to incubation with standard condition or 10 μM SAHA for 48 h. And then, PD-L1 knockdown in A549 cells was confirmed by ELISA.

Co-culture experiments

A549 cells (6×104 cells/well in 24-well plate) were transfected with the indicated concentration of immuno-DCD [siRNA] for 24 h prior to exposure to immuno-PCD [SAHA; 10 μM] for 48 h. Jurkat T-cells were activated with PMA and Ionomycin for 12 h and then co-cultured with A549 cells at effector to target (E-T) ratio of 10:1 for 48 h. Plates were washed with PBS twice and the living cells were stained with crystal violet solution for 5 min. After drying, the colonies were observed, and intensity was quantified.

Reverse-Transcription PCR

To evaluate activated T cells, Jurkat T-cells (1×106 cells/well in 6-well plate) were incubated with 50 ng/mL PMA and 1 μg/mL Ionomycin in a time-dependent manner. Total RNAs were isolated from Jurkat T-cells using Trizol reagent (Invitrogen Ltd., Paisley, Scotland) and phenol-chloroform separation protocol. And then, the reverse transcription-PCR (RT-PCR) assay was performed with cDNA synthesis kit (Solgent Co., Ltd, Korea). The primer sequences for human IL-2 were as follows: forward, 5'-ACCTCAACTCCTGCCACAAT-3'; reverse, 5'-GCACTTCCTCCAGAGGTTG-3'. The primer sequences for Human GAPDH used as an internal control, were as follows: forward, 5'-GTCAAGGCTGAGAACGGGAA-3'; reverse, 5'-AAATGAGCCCCAGCCTTCTC-3'. RT-PCR using the primers was performed according to the manufacturer's PCR condition. PCR products were electrophoresed on 1% agarose gels prepared in TAE buffer. Gel images were taken using a chemiluminescence analyzer (VilberLourmat, Eberhardzell, Germany).

Flow cytometric analysis

Expression of PD-1 protein in activated Jurkat T-cells was assessed by flow cytometry. Jurkat T-cells (1×106 cells/well in 6-well plate) were incubated with 50 ng/mL PMA and 1 μg/mL Ionomycin for 12, 24 and 48 h. Cells were incubated with anti-PD-1 antibody (1 μg/200 μL) (BioXcell, WestLebanon, USA) for 30 min at 4℃, washed three times, and incubated with FITC-labeled secondary antibody (Alexa Fluor 488; 1:1,000) for 30 min at room temperature. And then, the cells were washed three times in PBS and assessed by flow cytometry.

Cellular uptake and competitive binding assay

A549 cells (8×104 cells/well) were seeded in 24-well plate and incubated 24 h. DCD[siRNA] and immuno-DCD[siRNA] loaded with FITC labeled siRNA (50 pmole) were added to the A549 cells in serum-free or 60% serum-containing media for 4 h. The cells were washed twice with PBS and fixed with 4% paraformaldehyde. Fluorescence images were taken by fluorescence microscope. Also, a competitive binding assay was conducted using the anti-PD-L1 antibody to confirm antibody-mediated targeting. A549 cells (8×104 cells/well in 24-well plate) were pretreated with anti-PD-L1 antibody for 30 min at 4℃. before incubated with immuno-DCD[siRNA] for 4 h at 37℃. After incubation, the cells were harvested and collected with 200 μL of PBS analyzed by flow cytometry.

Cell binding analysis

A549 cells (8×104 cells/well) were seeded in 24-well plate and incubated for 24 h. The cells treated with PCD and immuno-PCD (20 μg) were incubated for 30 min in serum-free media. And then, the cells were washed twice with PBS and images were observed by fluorescence microscope. After that, the cells were collected with 200 μL of PBS and analyzed by flow cytometry.

Statistical analysis

Data are presented as means ± standard error (SD). Statistical significance of differences was examined by two-tailed Student's t-test method. The results with *P < 0.05 or **P < 0.01 were considered as statistically significant.

RESULTS

Anti-cancer effects of HDACi (SAHA) on cell viability and apoptosis of A549 cells

We assessed the anti-cancer effect of SAHA on cell viability and apoptosis in human non-small-cell lung cancer A549 cells. As shown in Fig. 1A, A549 cells were treated with SAHA at 1, 5, 10, 25, and 50 μM for 24 or 48 h. Cytotoxicity was evaluated using a WST assay. Cell growth was inhibited in a time- and concentration-dependent manner. The IC50 of SAHA in A549 cells was 10 μM after 48 h of incubation. Additionally, apoptosis was measured to determine whether SAHA induces apoptosis. A549 cells were incubated with SAHA in a concentration-dependent of SAHA for 48 h. The apoptosis assay was performed with Annexin V-FITC/PI staining. Fig. 1B and 1C show that the rate of total apoptosis demonstrated early and late apoptotic cells. This proportion of cells increased in a concentration-dependent manner. The cell viability and apoptosis assay results were similar. These results indicated that SAHA functions as a therapeutic agent by inducing cell growth inhibition and apoptosis in lung cancer cells.

Fig. 1. Anti-cancer effects of HDACi(SAHA) on tumor cells inhibition and apoptosis of A549 cells in 48 h. (A) A549 cells were treated with increasing dose (1~50 μM) of SAHA (vorinostat) for 24 or 48 h. The cell viability was assessed by WST assay. (B and C) A549 cells were treated with the indicated concentration of SAHA for 48 h, then Annexin V/PI assay was performed to measure the cell apoptosis. Bar charts showed quantitative data for total apoptosis rate at each the treated group. Data are presented as mean ± SD for triplicate experiments. A; ***P<0.001 compared with the control. C; ***P<0.001 compared with the control.

Induction of PD-L1 and chemokine expression by HDACi(SAHA) and PD-L1 siRNA in A549 cells

We performed ELISA to identify variations in PD-L1 and CCL5 levels induced by SAHA. Cells were treated with the indicated concentrations of SAHA for 24 and 48 h. As shown in Fig. 2A and 2B, PD-L1 and T-cell chemokine CCL5 expression levels were found to increase in a dose- and time-dependent manner. The number of cells treated with 10 μM SAHA was higher than that of the untreated control. These results demonstrate that SAHA can induce cancer resistance.

Fig. 2. Induction of PD-L1 and chemokine expression by HDACi (SAHA) and PD-L1 siRNA in A549 cells. (A and B) A549 cells were treated with increasing dose (1~50 μM) of SAHA for 24 or 48 h. (C and D) A549 cells were transfected with the indicated concentration of PD-L1 siRNA for 24 h and then treated with SAHA (10 μM) for 48 h. The levels of PD-L1 and chemokine (CCL5) were determined by ELISA. The data are presented as mean ± SD for triplicate experiments. A; **P<0.01 compared with the control. B; ***P<0.001 compared with the control. C; **P<0.01 compared with control, *P<0.05 compared with SAHA 10 μM group. D; *P<0.05 compared with control.

Furthermore, ELISA was used to evaluate whether the silencing effect of PD-L1 siRNA on PD-L1 expression was upregulated by HDACi. The cells were transfected with PD-L1 siRNA 100 and 200 nM for 24 h, followed by treatment with SAHA for 48 h. As resulted in Fig. 2C and 2D, we confirmed the elevation on PD-L1 expression is downregulated in a dose-dependent manner. PD-L1 siRNA treatment did not induce a decrease in chemokine expression. When treated with PD-L1 siRNA of 200 nM, PD-L1 expression was lower than that in the SAHA-treated control. These results demonstrate that PD-L1 siRNA effectively inhibited the upregulation of PD-L1 expression by SAHA.

PD-L1 targeted binding of immunoliposomes in A549 cells

To analyze the targeting effect of antibody-conjugated liposomes, we compared liposome-binding ability according to the presence or absence of the anti-PD-L1 antibody. First, we confirmed the stability of immunoliposomes in serum. DCD (DOTAP, cholesterol, DSPE-PEG2000) and Immuno-DCD loaded with FITC-labeled siRNA were transfected in serum-free or serum-containing media for 4 h, after which the cells were visualized by fluorescence microscopy. Under serum-free conditions, both DCD and immuno-DCD showed similar transfection efficiency to that of siRNA. While the transfection efficiency of immuno-DCD was effective in 60% serum conditions, DCD significantly affected siRNA delivery. As shown in Fig. 3A, the images demonstrated that immuno-DCD did not disrupt the delivery of siRNA by serum proteins. Hence, immuno-DCDs are more suitable for siRNA delivery than DCD.

Fig. 3. Intracellular localization and competitive binding assay of siRNA by anti-PD-L1 antibody conjugated lipoplexes. (A) A549 cells were incubated with DCD[siRNA] or immuno-DCD[siRNA] in serum-free or 60% serum-containing media. After 4 h, the intracellular localization of siRNA was taken by fluorescence microscope. (FITC-labeled siRNA (green), liposomes (red)) (B) Immuno-DCD [siRNA] was incubated to the A549 cells for 4 h at 37℃ after pre-treatment with the anti-PD-L1 antibody for 30 min at 4℃ and then the rhodamine of liposomes and FITC of siRNA control were analyzed by flow cytometry (Black: A549 control, Red: Immuno-DCD[siRNA], Green: anti-PD-L1 antibody + Immuno-DCD[siRNA]).

We also conducted a competitive binding assay to confirm the binding affinity by target specificity. As shown in Fig. 3B, the immuno-liposome group pretreated with anti-PD-L1 antibody showed significantly diminished shifting compared to the immuno-liposome alone group. The mean fluorescence intensity (MFI) of the red fluorescence of liposomes decreased 1.4-fold, and the transfection efficiency of siRNA decreased 1.6-fold. In addition, we measured the cell-binding capacity of the immuno-PCD liposomes using fluorescence imaging and flow cytometry. As shown in Fig. 4, immuno-PCD bound approximately 2-fold more than normal liposomes to A549 cells because of the effective recognition of PD-L1. These data verified that the liposome-conjugated PD-L1 antibody effectively recognize PD-L1 overexpressed cancer cells and enhances receptor-mediated internalization.

Fig. 4. PD-L1 targeted binding of immuno-PCD liposomes in A549 cells. (A) A549 cells were treated with Immuno-PCD for 30 min at 4℃ and then rhodamine in liposomes was visualized by fluorescent microscopy. (B) Cells were collected after trypsinization, and images of flow cytometry histograms were obtained (Black: A549 control, Green: PCD liposomes Red: Immuno-PCD liposomes).

PD-L1 protein silencing efficacy of immuno-DCD [siRNA] and cytotoxic effect of immuno-PCD[SAHA]

A549 cells were transfected with DCD or immuno-DCD loaded with 200 nM PD-L1 siRNA for 24 h and treated with SAHA (10 μM) for 48 h. We examined the efficiency of PD-L1 protein downregulation by using immunoliposomes. As shown in Fig. 5A, immuno-DCD liposomes delivered PD-L1 siRNA more effectively than DCD; hence, SAHA significantly inhibited the elevated expression of PD-L1. Additionally, we measured the anti-cancer effects of immunoliposomes loaded with SAHA. The cells were treated with PCD and immuno-PCD using SAHA at various concentrations. As shown in Fig. 5B, immuno-PCD[SAHA] showed a more significant growth inhibitory effect on A549 cells in a concentration-dependent manner than the control PCD [SAHA]. In summary, immunoliposomes delivered nucleic acids and drugs more effectively than control liposomes without antibody, demonstrating the anticancer effects of these formulations.

Fig. 5. PD-L1 protein silencing efficacy by immuno-DCD[siRNA] and cytotoxic effect by immuno-PCD[SAHA]. (A) A549 cells were incubated with PCD and immuno-PCD at the varied concentration of SAHA (5~15 μM) for 48 h. And then, cell viability was measured by WST assay. Error bars represent standard deviation of three independent experiments. *P<0.05 compared with PCD[SAHA] and Immuno-PCD[SAHA]. (B) A549 cells were incubated with PCD and immuno-PCD at the varied concentration of SAHA (5~15 μM) for 48 h. The cell viability was measured by WST assay. Error bars represent standard deviation of three independent experiments. A; ***P<0.001 compared with SAHA 10 μM group, **P<0.01 compared with DCD[siRNA] group. B; *P<0.05 compared with PCD[SAHA] and Immuno-PCD[SAHA].

Expression of PD-L1 and chemokines by cocktail treatment with immuno-DCD[siRNA] and immuno-PCD[SAHA]

As shown in Fig. 6, Chemokine and PD-L1 expression levels with immunoliposomes were compared to investigate the synergistic effect of the cocktail treatment with immuno-DCD[siRNA] liposomes and immuno-PCD[SAHA]. Immuno-DCD [siRNA; 200 nM] was added for 24 h, followed by immuno-PCD [SAHA; 10 μM] for 48 h. Thereafter, changes in PD-L1 and CCL5 levels were evaluated by ELISA. Similar trends are shown in Fig. 2C and 2D, and the combination of the two immuno-liposomes also showed effective inhibition of PD-L1 expression. This indicated that the immune liposomes were taken up by targeting the PD-L1 protein in the cell. In addition, CCL5 expression by cocktail treatment was shown to be more effective than immuno-PCD [SAHA] treatment alone.

Fig. 6. Expression of PD-L1 and chemokine by cocktail treatment of immuno-DCD[siRNA] and immuno-PCD[SAHA] liposomes. A549 cells were incubated with immuno-DCD[siRNA] for 24 h and then treated with immuno-PCD[SAHA] (10 μM) for 48 h. The expression levels of (A) PD-L1 and (B) chemokine (CCL5) were determined by ELISA. The data are presented as means ± SD for triplicate experiments. A; *P<0.05 compared with immuno-PCD[SAHA]. B; ***P<0.001 compared with control, *P<0.05 compared with control.

Effect of reduced PD-L1 expression on T cell-mediated A549 cell death

To investigate the interaction with PD-1 in T cells following changes in PD-L1 expression in tumor cells, A549 cells were co-cultured with Jurkat-T cells expressing PD-1. Phorbol myristate acetate (PMA) and ionomycin are used to induce T cell proliferation and activation. Jurkat T cells were activated with PMA (50 ng/mL) and ionomycin (1 μg/mL) over time to induce optimal T cell activation and PD-1 expression. Therefore, after measuring the expression level of cytokine IL-2, which is expressed when activated T cells proliferate, we confirmed the cancer cell suppression effect through co-culture with T cells and A549 cancer cells. As shown in Fig. 7A, we confirmed the expression of IL-2, which is related to T cell proliferation, by RT-PCR. Jurkat-T cells activated by PMA and ionomycin led to the highest IL-2 expression at 12 h. IL-2 expression gradually decreased at 24 and 48 h. PD-1 expression levels also showed a similar trend in Fig. 7B, and optimal activation was observed after 12 h. A549 cells were treated with immuno-PCD[SAHA; 10 μM] alone or with 100 nM or 200 nM of PD-L1 siRNA in immuno-DCD[siRNA] transfection for 24 h, and the cells were co-cultured with activated Jurkat-T cells with effector to target ratios of 10:1 for 48 h. In Fig. 7C, the bar charts show the quantitative data for surviving cancer cells stained with crystal violet. A549 cell death was effectively induced in the immuno-PCD[SAHA; 10 μM] and immuno-DCD[siRNA; 200 nM] co-treated group, and the surviving cancer cells showed a relative difference of 50% compared to the only immuno-PCD[SAHA; 10 μM] group. Finally, these results demonstrated that the inhibition of PD-L1 expression in cancer cells by PD-L1 siRNA induces T cell-mediated killing via the PD-1/PD-L1 interaction. Finally, this result confirmed that siRNA did not interfere with chemokine expression and demonstrated that the cocktail treatment of SAHA and PD-L1 siRNA is an effective nanocarrier capable of inducing T cells.

Fig. 7. PD-1 expression in Jurkat T cells and interaction of PD-1/PD-L1 interaction in Jurkat cell-mediated A549 cell killing assay. Jurkat T cells were incubated with or without PMA and Ionomycin for up to 48 h. (A) The mRNA level of IL-2 was determined by RT-PCR. (B) Expression of PD-1 receptor was determined using a flow cytometry. (C) A549 cells were transfected with or without immuno-DCD [siRNA; 100 nM or 200 nM] for 24 h prior to exposure to immuno-PCD[SAHA; 10 μM] for 48 h before co-incubation with activated PD-1 expressing Jurkat T cells with effector to target ratios of 10:1. After 48 h of incubation, remaining cancer cells in plate were stained by crystal violet. C; **P<0.01 compared with to immuno-PCD[SAHA] only.
DISCUSSION

Immune therapies have been developed using immune checkpoint inhibitors, antibodies, vaccines, and immune cell therapy. Immune checkpoint targeted therapies on the PD-1/PD-L1 pathway have shown substantial effects in the treatment of a variety of cancers using antibodies that block the pathway interaction (Xu et al., 2018). Recently, numerous studies on lung cancer have reported that combination immunotherapy can lead to better outcomes than monotherapy (Esteva et al., 2019; Yu et al., 2019). Anti-cancer agents used for immunotherapy effectively induce T-cell recruitment and activity in the tumor microenvironment (Zheng et al., 2016). Among the anti-cancer agents, we chose HDAC inhibitors and screened them with SAHA as the focus. First, cytotoxicity and apoptosis assays were conducted to assess the anti-cancer effect of SAHA on human non-small-cell lung cancer A549 cells. The results demonstrated that SAHA exerted anti-proliferative and apoptotic effects on the cells (Fig. 1). This anti-cancer agent also induces T cell recruitment in the lung cancer cell line A549. This proved that SAHA is a promising drug that can lead to active immunity as well as the direct death of cancer cells. Several studies have shown that HDAC inhibitors cause overexpression of PD-L1 in tumor cells, which can result in immune invasion of tumor cells (Woods et al., 2015). In this regard, we confirmed the levels of PD-L1 induced by SAHA, which showed a concentration-dependent increase.

This study aimed to prevent PD-L1 overexpression in tumor cells by SAHA using gene silencing through RNA interference therapy, which may induce effective immunotherapy by blocking the PD-1/PD-L1 pathway. According to the siRNA concentration, PD-L1 siRNA inhibited the increase in PD-L1 levels induced by SAHA. In addition, the effect of PD-L1 expression using control siRNA was insignificant. To confirm the effect of siRNA-induced reduction in PD-L1 expression on chemokine induction, we evaluated CCL5 expression. Surprisingly, the combination of PD-L1 siRNA with SAHA did not offset the chemokine expression induced by SAHA and showed similar expression rates as SAHA alone (Fig. 2). These results confirm that after combination therapy of siRNA and SAHA, this siRNA can effectively suppress PD-L1, which can be increased by SAHA, and increase the expression of T cell-induced cytokines. This is thought to be the result of showing the possibility of inducing active immunity.

Many studies have indicated that tumor cells inhibit T cell proliferation when PD-L1 overexpressing tumor cells are co-cultured with T cells in a cell-to-cell contact manner. Accordingly, Jurkat T cell-mediated cell killing assay was performed with SAHA- and PD-L1 siRNA-treated groups to confirm the interaction with PD-1 expressing Jurkat-T cells by PD-L1 expression. Jurkat-T cells treated with SAHA suppressed IL-2 expression, which is involved in T cell activity and proliferation (Coombs et al., 2016). The increase in PD-L1 expression by SAHA was diminished by PD-L1 siRNA, which reduced the interaction with PD-1 in T cells, thereby inducing effective T cell-mediated cell death (Fig. 7).

In current cancer treatment, drug delivery systems have been continuously researched to overcome chemotherapeutic drawbacks, such as inducing systemic toxicity (Alavizadeh et al., 2016). Similarly, many carriers have been developed to deliver siRNAs for gene therapy used in cancer therapy. Among the delivery systems, nanocarriers, including liposomes, are promising carriers for cancer treatment (Kim et al., 2017; Song et al., 2016). They can be used to easily modify surfaces and encapsulate hydrophilic and hydrophobic compounds during drug delivery (Nele et al., 2019). However, liposomes can be easily eliminated by the reticuloendothelial system (RES) during circulation in the body. PEG is indispensable for coating conventional liposome formulations to reduce the recognition of foreign substances and prolong the circulation time (Huwyler et al., 1996).

In this study, we designed liposomes conjugated with antibodies to achieve superior binding to PD-L1 in cancer cells. SAHA was encapsulated in POPC-based liposomes (PCD liposomes) and PD-L1 siRNA was complexed with DOTAP-based liposomes (DCD liposomes) using characteristic electrostatic bonding. We compared the binding effects of immunoliposomes with those of planar liposomes on A549 cells. Immunoliposomes showed enhanced targeting by inducing PD-L1 expression in A549 cells (Fig. 3 and 4). Immuno-DCD liposomes[siRNA] confirmed the silencing effect of PD-L1 expression; it showed insignificant downregulation of PD-L1 protein compared to control liposomes. Next, the combination of immuno-PCD[SAHA] liposomes and immuno-DCD[siRNA] was used to evaluate the anticancer effect using a cytotoxicity assay. Cell growth was inhibited more effectively than PCD[SAHA] liposomes alone in a concentration-dependent manner (Fig. 5). The combination of the two immunoliposomes was demonstrated through the expression of PD-L1 and chemokine CCL5. The expression of CCL5 by immuno-PCD[SAHA] was increased more than 2-fold compared to that by free SAHA in Fig. 2, which proved that the delivery of drugs and nucleic acids using immunoliposomes was quite useful (Fig. 6).

In summary, liposomes have been used to enhance therapeutic effects, while compensating for the shortcomings of free drugs and gene delivery. In this study, two immunoliposomes in which an anti-PD-L1 antibody was conjugated to liposomes with SAHA and PD-L1 siRNA were developed to specifically target PD-L1-expressing tumor cells based on the characteristics of these liposomes. Both immunoliposomes were internalized in A549 lung cancer cells by recognizing PD-L1, and the combination of these liposomes effectively led chemokine CCL5 expression. Therefore, these agents can be used for anticancer treatment by combining immunotherapeutic agents and targeted therapies and present a new paradigm for cancer treatment as new carriers.

Abbreviations

C-C motif chemokine ligand 5, CCL5; histone deacetylase inhibitor, HDACi; non-small cell lung cancer, NSCLC; suberoylanilide hydroxamic acid, SAHA; programmed death-ligand 1, PD-L1.

ACKNOWLEDGEMENT

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF-2021R1F1A1062932).

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

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