Introduction
Cancer immunotherapy has emerged as a revolutionary approach in the treatment of various malignancies, offering significant promise for long-term disease control and survival benefits [ 1 ]. Among the diverse immunotherapeutic strategies, the modulation of the immune system to recognize and eliminate tumor cells has shown remarkable efficacy [ 2 ]. One such promising strategy involves the utilization of bone marrow-derived dendritic cells (BMDCs) as cellular vectors for delivering therapeutic payloads directly into the immune system [ 3 ]. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a pivotal role in initiating and shaping adaptive immune responses. Adoptive DC vaccines employ the patient's own DC cells and specific antigens, thereby ensuring high safety and minimizing the risk of eliciting rejection responses [ 4 ]. This vaccine boasts broad applicability, being suitable for various tumor types and unconstrained by MHC Class I antigens [ 5 ]. Consequently, it holds promising prospects as a significant research direction in the field of tumor immunotherapy for widespread application. Currently, DCs-based cellular therapy has achieved remarkable outcomes in clinical trials involving various tumor types, including breast cancer, multiple myeloma, prostate cancer, renal cell carcinoma, malignant melanoma, colorectal cancer, and non-small cell lung cancer [ 6 , 7 ]. Notably, the U.S. Food and Drug Administration (FDA) has approved Sipuleucel-T as the first DC vaccine for the treatment of prostate cancer [ 8 ]. Despite significant advancements in adoptive DC vaccination, the efficient loading of tumor antigens and the activation status of DCs remain pivotal issues in the development of adoptive DC-based vaccines [ 9 , 10 ].
By harnessing the antigen-presenting capabilities of BMDCs, researchers have explored ways to enhance their immunogenicity against tumor antigens, thereby stimulating robust antitumor immune responses [ 11 ]. One innovative approach involves the transfection of BMDCs with messenger RNA (mRNA) encoding specific tumor antigens [ 12 ]. This method allows for the in vivo expression of these antigens, which can then be presented to T cells, activating antitumor immunity. This strategy also circumvents numerous risks and challenges associated with direct injection of mRNA vaccines, such as hepatotoxicity and other toxic side effects [ 13 ]. The activation status of DCs encompasses several pathways. When pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) are recognized by pattern recognition receptors (PRRs) on DCs, it triggers the activation or maturation of these cells. These PRRs include surface and endosomal Toll-like receptors (TLRs), C-type lectins, and cytosolic sensors [ 14 , 15 ]. Furthermore, downregulating the expression of programmed death-ligand 1 (PD-L1) on DCs can relieve the inhibitory effect of PD-1 on T cells, thereby enhancing T-cell activation, promoting their proliferation and differentiation into effector T cells, and ultimately augmenting the cytotoxic effect on tumor cells [ 16 ]. The improved antigen presentation efficiency of DCs contributes to bolstering T-cell immune responses and further enhancing and modulating the body's antitumor immune reactions [ 17 ].
However, despite the promising results of antigen-specific immunotherapy, its efficacy is often limited by immune evasion mechanisms employed by tumor cells [ 18 ]. One such mechanism involves the expression of programmed death-ligand 1 (PD-L1) on tumor cells, which interacts with its receptor PD-1 on T cells to inhibit their activation and proliferation, thereby promoting immune escape [ 19 , 20 ]. To overcome this challenge, strategies to block the PD-1/PD-L1 interaction have been developed, including the use of anti-PD-L1 antibodies [ 21 , 22 ]. Monoclonal antibodies against PD-L1 can specifically bind to PD-L1 on the surface of tumor cells, thereby blocking the binding between PD-L1 and PD-1 on the surface of T cells. This blocking action effectively relieves the inhibition of T cells by the PD-L1/PD-1 pathway [ 23 ]. Once the binding between PD-L1 and PD-1 is blocked, T cells are no longer suppressed, leading to enhanced proliferative capacity [ 24 , 25 ]. Concurrently, the ability of T cells to secrete cytokines, which play crucial roles in immune responses, is also improved [ 26 ]. Therefore, altering the tumor immune tolerance microenvironment through immune checkpoint blockade holds promise for enhancing the antitumor immunotherapy efficacy of tumor vaccines.
Our approach involves the co-loading of LNPs with mRNA encoding ovalbumin (OVA) model tumor antigen and siRNA targeting PD-L1 by microfluidic control technology. The LNP/mOVA/siPD-L1 complexes are then used to transfect BMDCs, resulting in the expression of the tumor antigen and the silencing of PD-L1 expression. Following transfection, the genetically modified BMDCs are reintroduced into mice, where they are expected to stimulate potent antitumor immune responses. Additionally, to further enhance the therapeutic effect, we administer anti-PD-L1 antibodies to block PD-1/PD-L1 interactions ( Fig. 1 ). The objective of this study is to evaluate the therapeutic potential of this combined immunotherapy approach in a preclinical mouse colorectal cancer tumor model. We hypothesize that the dual modulation of the immune system through the expression of tumor antigen mRNA and silencing of PD-L1 expression in BMDCs, and blockade of the PD-1/PD-L1 interaction with anti-PD-L1 antibodies will lead to enhanced antitumor immunity. Our findings have the potential to pave the way for the development of more effective and personalized immunotherapeutic strategies for the treatment of cancer, ultimately improving patient outcomes and survival rates.
Materials and methods
Materials
Heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM102), 1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Cholesterol, lipopolysaccharide (LPS), 1.2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), murine interleukin4 (IL-4), 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT), carboxyfluorescein succinimidyl ester (CFSE), and murine granulocyte-macrophage colony-stimulating factor (GM-CSF) were sourced from Sigma-Aldrich (St. Louis, MI, USA). Enhanced green fluorescence protein mRNA (EGFP mRNA), ovalbumin mRNA (OVA mRNA), and Cy5-mRNA were acquired from TriLink BioTechnologies (California, USA). Quant-iT RiboGreen RNA quantify kit and Lipofectamine™ 3000 were obtained from Thermo Fisher Scientific (Massachusetts, USA). An ultrafiltration centrifugal tube with a molecular weight cut-off (MWCO) of 100 kDa was procured from Millipore (USA). PD-L1 siRNA (siPD-L1, 5′-GAGGUAAUCUGGACAAACATT-3′) and FAM-siNC were purchased from GenePharma (Shanghai, China). ELISA Kits for IL-6, IL-12p70, TNF-α, and IFN-β were sourced from ABclonal (Wuhan, China). An anti-PD-L1 recombinant monoclonal antibody was acquired from BioXcell, Inc. (West Lebanon, USA). All cell culture reagents were procured from Gibco (California, USA). Additionally, all other flow fluorescent antibodies were ordered from eBioscience (California, USA).
Preparation of LNP/mOVA/siPD-L1 nanoparticles
The LNP/mOVA/siPD-L1 nanoparticles were prepared by rapidly mixing an ethanol solution of lipids and an aqueous solution of mRNA payloads using a microfluidic technique. The ethanol solution comprised of SM102, cholesterol, DSPC, and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5 in ethanol [ 27 ]. The aqueous solution was prepared by diluting OVA mRNA and siPD-L1 in 0.05 mM RNase-free sodium citrate buffer. The optimized mass ratio of mRNA and siRNA was 5:1. Subsequently, the LNP/mOVA/siPD-L1 nanoparticles were prepared via a microfluidic equipment (Aitesen, China). The ethanol phase and aqueous phase were blended at a volume ratio of 1:3, and performed flowing rates at 4 mL/min and 12 mL/min, respectively [ 28 ]. The non-encapsulated payloads were removed by ultrafiltration tubes at 10000× g . The resulting LNP/mOVA/siPD-L1 nanoparticles were achieved and stored at 4 °C.
Characterizations of LNP/mOVA/siPD-L1 nanoparticles
The content of mRNA within nanoparticles was determined by a Quant-iT RiboGreen RNA kit and its encapsulation efficiency was calculated by referencing the standard curve via the following equation [ 29 ]: mRNA EE%=(Total mRNA-Unencapsulated mRNA)/(Total mRNA) × 100.
The particle sizes, polydispersity indexes (PDI), and zeta potentials for LNP/mOVA/siPD-L1 nanoparticles were determined by dynamic light scattering (Malvern, Zetasizer APS, UK) at a constant mRNA concentration of 5 μg/mL [ 30 ]. Furthermore, the surface morphology of the nanoparticles was observed using a scanning electron microscopy (SU7000, Hitachi, Japan). To assess the storage stability of LNP/mOVA/siPD-L1 nanoparticles, freshly prepared nanoparticles were subjected to conditions at 4 °C, and their particle sizes, zeta potentials, and drug encapsulation efficiencies were measured on days 0, 1, 2, 3, 4, 5, 6, and 7.
Biological functions of LNP/mOVA/siPD-L1 nanoparticles
Biocompatibility study in BMDCs
The male BALB/c mice (6 weeks old, 18–20g) were purchased from Charles River Company (Beijing, China) and fed under the guidance of the Animal Care and Use Committee of Jilin University (SMP-ZL-002/BG01). BMDCs were isolated and extracted from the femurs of BALB/c mice. The single cells were then cultured in RPMI 1640 medium supplemented with 10 % FBS, 20 ng/mL GM-CSF, 10 ng/mL IL-4, and 1 % penicillin-streptomycin for 7 days and the medium were half changed every two days. For biocompatibility study, the cells were seeded into 96-well plates at a density of 0.8 × 10 3 cells per well. LNP/mOVA/siPD-L1 solutions were added to the wells at an 0.4 μg/mL equivalent dose of mRNA concentrations. After a 24-h incubation period, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well. After a further 4 h, the medium was gently discarded, and 150 μL of DMSO was added [ 31 ]. Subsequently, the absorption value at 490 nm was measured using a microplate reader. The cell viability was calculated according to the manufacturer's protocols.
Intracellular uptake in BMDCs
Immature BMDCs were seeded into laser confocal dishes, maintaining a cell density of 5.0 × 10 5 cells per dish. Following an overnight incubation, LNP/Cy5-mRNA/FAM-siRNA nanoparticles were added into the wells and incubated for 4 h, ensuring a final Cy5-mRNA concentration of 0.5 μg/mL. The BMDCs were gently washed with PBS and fixed with 4 % paraformaldehyde for 15 min. The nuclei were then stained with DAPI for 5 min under dark conditions [ 32 ]. After washing with PBS, the samples were observed under confocal laser scanning microscopy (CLSM, ZEISS LSM780, Germany).
Gene transfection in BMDCs
The mRNA transfection experiments were optimized by EGFP mRNA encoding the green fluorescent protein gene and PD-L1 siRNA [ 33 ]. Briefly, BMDCs were plated in 24-well plates at a density of 5.0 × 10 5 cells per well, and incubated overnight. Subsequently, LNP/EGFP mRNA/siPD-L1 nanoparticles with various mass ratios were added into the wells and incubated for 24h. For EGFP detection, the transfected cells were observed under an inverted fluorescent microscope (TI-S, Nikon, Japan). Subsequently, the cells were collected, centrifuged at 1000 rpm for 3 min, and washed three times with cold PBS buffer. Finally, the harvested cells were labelled with PE labelled anti-PD-L1 and analyzed using a flow cytometer, and the data were processed through Flowjo software.
Dual-bioengineering of BMDCs
BMDCs were seeded in 10 cm dishes at a density of 5.0 × 10 6 cells per dish, and incubated overnight. Subsequently, LNP/mOVA/siPD-L1 nanoparticles with 5 μg of OVA mRNA were added into the dishes and incubated for 24h. The suspension cells were collected and counted using the cell counter and transferred to a sterile Eppendorf tube for s.c. administration.
Immunomodulatory effect of dual-bioengineered BMDCs
The immunomodulatory effects of LNP/mOVA/siPD-L1 nanoparticles were evaluated in BMDCs [ 34 ]. BMDCs were seeded into 24-well plates at a density of 5.0 × 10 4 cells per well and incubated overnight. Subsequently, PBS, OVA mRNA and siPD-L1, LNP/mOVA/siPD-L1 nanoparticles, and LPS were added to the wells and incubated for another 24 h. The final concentration of mRNA was maintained at 0.5 μg/mL. The BMDCs were harvested, washed with PBS, and labelled with fluorescence labelled anti-CD11c, anti-CD80, and-CD86, and anti-MHC II antibodies. All the fluorescent conjugated antibodies were shown in Table S1 . Additionally, the antigen cross-presentation ability of the BMDCs was assessed by measuring the percentage of SIINFEKL-H2k + cells within the CD11c + cell population using a flow cytometer. The collected data were further analyzed using FlowJo software. Furthermore, the supernatants were evaluated for the secretion of cytokines, including interleukin-12p70 (IL-12p70), interleukin-6 (IL-6), tumor necrotic factor (TNF-α), and interferon-β (IFN-β) using ELISA kits ( Table S2 ).
T cell activation by the dual-bioengineered BMDCs
BMDCs were subjected to a 24-h incubation period with three different treatments: PBS, a combination of OVA mRNA and siPD-L1, and LNP/mOVA/siPD-L1 nanoparticles. Throughout this incubation, the mRNA concentration was consistently maintained at 0.5 μg/mL. Subsequently, CD8 + T cells were isolated from single-cell suspensions of splenocytes derived from OT1 transgenic mice, utilizing the Mouse CD8a Positive Selection Kit II (StemCell). These isolated T cells were then stained with CFSE dye for tracking purposes. Next, the BMDCs that had been pulsed with LNP/mOVA/siPD-L1 were co-cultured with the stained OT1 CD8 + T cells for a duration of three days. Finally, a fluorescence-activated cell sorting (FACS) assay was conducted to evaluate the proliferation index of the CD8 + T cells [ 10 ].
Construction of OVA stable expressing colorectal cells (CT26-OVA)
A stable OVA-expressing CT26-OVA cell line derived from colorectal cancer cells CT26 was successfully established through lentiviral infection [ 34 ]. Briefly, 293T cells served as the host cells for lentiviral particle packaging, and were co-transfected with the three-plasmid system comprising pLVs-CMV-OVAL-3xFLAG-Puro, pSPAX2, and pMD2.G using Lipofectamine™3000, at a mass ratio of 2:1.5:1, with a final concentration of 0.25 μg/mL for pMD2.G. Six hours post-transfection, the medium was replaced and incubation continued for an additional 42 h. The resultant lentiviral particles were harvested and purified using a viral concentration kit. The lentiviral supernatant was then added to CT26 cells at a 1:1 vol ratio with DMEM medium, and the cells were co-incubated for 24 h before replacing the supernatant with fresh lentiviral supernatant. To further purify the CT26-OVA cells, they were transferred to fresh medium containing puromycin (2 μg/mL), with medium exchanges every 2–3 days. After a week of cultivation, the CT26-OVA cells were successfully harvested.
Anti-tumor therapy using dual-bioengineered BMDCs as prophylactic tumor vaccines
BALB/c mice (male, 6 weeks) were purchased from Charles River Company (Beijing, China) and fed for a week to acclimate. From day −23 to day −16, the dual-bioengineered DCs were harvested by transfecting with LNP/mOVA/siPD-L1 nanoparticles and injected subcutaneously into the groin region with 1.0 × 10 5 cells suspended in 50 μL of PBS. Un-transfected DCs and PBS group were used as the control groups. On the -8th and -1st days, the second and third booster immunizations were administered, respectively. Subsequently, CT26-OVA cells were injected subcutaneously into the dorsal region of the mice with a concentration of 5.0 × 10 5 cells per mouse on day 0. Tumor sizes and body weights were monitored twice a day, and tumor volumes were calculated using the formula (length × width 2 )/2 [ 10 ]. For survival experiment, according to animal ethical requirements, when the tumor volume of mice exceeds 1500 mm 3 , it is defined as the death of mice.
Anti-tumor therapy using dual-bioengineered BMDCs as therapeutic tumor vaccines
BALB/c mice (male, 6 weeks) were first subcutaneously injected with CT26-OVA cells with a concentration of 5.0 × 10 5 cells (100 μL per mouse, suspended in PBS) per mouse into the dorsal region of the mice on day 0. On the 9th day, the tumor bearing mice were randomly divided into 3 groups, 5 mice in each group, to receive subcutaneous immunization with the dual-bioengineered DC vaccines, and two booster immunization sessions were given on the 13th and 17th day. Un-transfected DCs and PBS group were used as the control groups. Tumor sizes and body weights were monitored twice a day. On the 20th day, when the tumor volume of mice in the control group exceeded 1500 mm 3 , all the mice were euthanized, the inguinal lymph nodes (LNs), spleens, and the tumors were surgically excised and digested into a single-cell suspension. The quantification of activated DCs (CD11c + CD86 + ) in LNs, memory CD8 + T cells (CD3 + CD8 + CD44 + CD62L − ) in spleens, and various immune cells within the tumors was conducted, including CD8 + T cells (CD3 + CD8 + ), CD4 + T cells (CD3 + CD4 + ), M1 cells (CD11b + CD8 + F4/80 + ), and Treg cells (CD4 + CD206 + FoxP3 + ) [ 35 ]. The mean fluorescence intensity (MFI) of PD-L1 expression on tumor cells was detected using a flow cytometer and analyzed via FlowJo software.
For immunofluorescence staining, the tumor tissues were meticulously prepared into paraffin slices. These slices underwent a series of processing steps, including dewaxing paraffin, antigen retrieval, circle drawing, hydrogen peroxide blocking, serum blocking, primary antibody incubation including anti-CD4, anti CD8, and anti-PD-L1, secondary antibody incubation, DAPI staining of cell nuclei, autofluorescence quenching, and mounting [ 36 ]. Primary antibodies for immunofluorescence staining were listed in Table S3 . The resulting slices were observed using a fluorescence microscope (Zeiss 780, Germany), revealing crucial information about the cellular interactions and expression patterns.
To further examine the expression of CD4, CD8, and Ki67, immunohistochemistry and Masson staining were conducted. Paraffin-embedded tumor sections were permeabilized and incubated with primary antibodies against CD4, CD8, and Ki67, after which the immunohistochemical staining protocol was followed. For Masson staining, paraffin-embedded tumor slides were stained in accordance with the instructions provided by the Masson staining kit (Solarbio, Beijing, China).
Security analysis
The biomarkers present in the serum were quantified using ELISA [ 37 ]. In brief, the serum samples were separated via centrifugation at 3000 rpm for 20 min. Subsequently, these samples were employed to detect interleukin-6 (IL-6), interleukin-12 (IL-12p70), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), creatinine (CRE), blood urea nitrogen (BUN), and uric acid (UA) levels.
The hematological analysis was conducted to assess the acute side effects and toxicities associated with various treatments. The blood samples were meticulously analyzed, encompassing white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) levels, hematocrit (HCT) values, and platelet (PLT) counts.
The histological features of tumors and major organs, including the heart, liver, spleen, lung, and kidney, were meticulously stained with hematoxylin and eosin (H&E) [ 21 ]. The prepared slices were then examined through an upright microscope (DS-Ri2, Nikon, Japan), affording us detailed insights into the cellular structure and morphology.
Combination therapy of anti-PD-L1 and dual-bioengineered BMDCs in the CT26-OVA therapeutic model
BALB/c mice (male, 6 weeks) were first subcutaneously injected with CT26-OVA cells with a concentration of 5.0 × 10 5 cells per mouse into the dorsal region of the mice on day 0. On the 9th day, the tumor bearing mice were randomly divided into 4 groups, 5 mice in each group, to receive subcutaneous immunization with the dual-bioengineered DC vaccines and intraperitoneal injection of anti-PD-L1 antibody, and two booster immunization sessions were given on the 13th and 17th day. Anti-PD-L1 antibody, dual-bioengineered DCs, and PBS group were used as the control groups. Tumor sizes and body weights were monitored twice a day [ 38 ]. For survival experiment, according to animal ethical requirements, when the tumor volume of mice exceeds 1500 mm 3 , it is defined as the death of mice.
Next, the immune memory effect of the combination treatment strategy was investigated by the tumor re-challenge experiment. Briefly, on day 45, the mice were rechallenged with CT26-OVA cells (5.0 × 10 5 cells/100 μL) in the contralateral dorsal region of the mice, and the 2nd tumors were measured until day 66. Tumor sizes were monitored twice a day and the tumor volume was calculated using the formula: (length × width 2 )/2.
Statistical analysis
GraphPad Prism 8.0 software was used to analyze data and create graphs. The statistical significance was calculated by Student's t-test. For the statistical significance of survival curve, we used Log-rank (Mantel-Cox) test.
Results and discussion
Preparation and characterization of LNP/mOVA/siPD-L1 nanoparticles
DC vaccines represent a novel class of vaccines that harness dendritic cells loaded with tumor cells, tumor antigen peptides, or genes expressing tumor antigens as antigen-presenting cells (APCs) to induce antitumor immune responses [ 39 ]. DC cells are the most potent specialized APCs known in humans for activating T cells, efficiently conveying antigen information to T lymphocytes and activating adaptive immune responses, thereby recognizing and attacking abnormal cells within the body [ 4 ]. DC vaccines exhibit high safety profiles, as they utilize the patient's own DC cells, which are activated and expanded ex vivo before being reintroduced into the patient, thereby minimizing immunogenicity and cytotoxicity [ 40 ]. Furthermore, DC vaccines can persistently mobilize various cytokines in vivo to participate in tumor cell clearance while reducing damage to normal cells, ensuring exceptional safety. Additionally, DC vaccines possess memory, enabling them to exert their effects for decades. Tumor mRNA vaccines not only activate humoral and cellular immunity through antigen proteins but also leverage the immunostimulatory properties of mRNA itself to induce more potent immune responses [ 41 ]. In the field of oncological treatment, tumor mRNA vaccines demonstrate multiple advantages, including high efficiency, personalization, safety, rapid response and large-scale production capabilities, dual immune mechanisms and flexibility, as well as long-term benefits [ 42 ]. These advantages position tumor mRNA vaccines as a promising new immunotherapy with broad prospects.
In this study, the lipid nanoparticles were formulated with SM102, DSPC, Cholesterol, and DMG-PEG2000 in their molar ratios of 50:10:38.5:1.5 in ethanol solution. OVA mRNA encoding ovalbumin model antigen was selected for treating CT26-OVA tumors. In addition, siPD-L1 was used to further improved the immune activation of DCs. The LNP/mOVA/siPD-L1 nanoparticles were formulated via a microfluidic control technology ( Fig. 2 A). The encapsulation rate of nucleic acid is approximately 92.5 %. Suitable particle size and dispersibility are crucial for nucleic acid delivery efficacy [ 43 ]. As shown in Fig. 2 B, LNP/mOVA/siPD-L1 nanoparticles demonstrated a homogenous distribution and uniform dispersion by dynamic light scattering and SEM. It was evident that these LNPs had a remarkably uniform particle size distribution, with an average diameter precisely around 80 nm. The well-defined and highly homogeneous morphology of the LNPs in the SEM micrograph was a strong indicator of the excellent reproducibility and controllability of the preparation process. To verify the stability and reproducibility of the preparation process, we supplemented the preparation with three batches of LNP/mRNA/siRNA nanoparticles and measured their particle size, PDI, and surface potential. The results also demonstrated that the particle sizes and zeta potentials of the nanoparticles prepared in the three batches were comparable, further proving the stability of the preparation process ( Table S4 ). In addition, the stability of the nanoparticles was detected by storing them under 4 °C for 7 days. During the storage process, the particle sizes of the nanoparticles was always stable at 75–85 nm, and their PDI value are less than 0.1 ( Fig. 2 C). In addition, the surface potentials of the nanoparticles were always kept in a slightly negative state, and there was no significant change ( Fig. S1 ). The drug encapsulation rate remained above 90 %, which further demonstrated the storage stability of LNP/mOVA/siPD-L1 nanoparticles ( Fig. S2 ).
Studying the cellular activity of DC vaccines prior to reinfusion ensures that the DC cells maintain their antigen-presenting function upon reinfusion, effectively activating immune cells such as T cells and triggering a robust immune response. Consequently, we employed the MTT assay to assess the viability of dual-bioengineered BMDCs after gene transfection. The results demonstrated ( Fig. S3 ) that the survival rate of transfected cells exceeded 90 %, which further guarantees the efficacy and safety of the dual-bioengineered DCs. Efficient delivery of exogenous genes into bone marrow-derived dendritic cells (BMDCs) is a prerequisite for achieving high transfection efficiency. We utilized microfluidic technology to synthesize LNP/FAM-siRNA/Cy5-mRNA nanoparticles and co-incubated them with BMDCs for 4 h, subsequently observing cellular endocytosis using CLSM. As shown in Fig. 2 D, both distinct green and red fluorescence were observed within the BMDCs, indicating that the LNP effectively delivered both siRNA and mRNA into the cells. Next, we further investigated the transfection efficiency of mRNA within BMDCs. As depicted in Fig. 2 E, the green fluorescence colocalized with BMDCs, confirming the high transfection efficiency of EGFP mRNA within the cells. Flow cytometry results ( Fig. 2 F) demonstrated that the LNP/mOVA/siPD-L1 group significantly reduced the expression of PD-L1 protein on the cell surface, indicating the potent gene silencing effect of siPD-L1. Downregulation of PD-L1 could block the interaction between DCs and T lymphocytes, further promoting the activation of T lymphocytes.
In vitro immune stimulation of LNP/mRNA/siRNA nanoparticles
Under in vitro conditions, BMDCs were activated through a dual-gene transfection approach involving mRNA and siRNA, simultaneously enabling the presentation of tumor antigens. Upon reinfusion, these BMDCs continued to exert their antigen-presenting function in vivo by presenting tumor antigens to T cells, thereby activating T-cell-mediated immune responses against tumors. In this study, we co-incubated LNP/mOVA/siPD-L1 nanoparticles with BMDCs for 24 h. Subsequently, the maturation and cross-presentation of antigens by BMDCs, as well as the secretion of cytokines in the culture medium were detected ( Fig. 3 A). As shown in Fig. 3 B–D, mOVA/siRNA group demonstrated a negligible difference compared with PBS control group, while the cell maturation rate of LNP/mOVA/siPD-L1 nanoparticles showed significant improvement, almost comparable to that of the LPS treated group.
Efficient antigen cross-presentation significantly boosts the activation and responsiveness of cytotoxic T cells, marking a crucial stage in tumor immunotherapy [ 32 ]. By stimulating potent cellular immune responses, vaccines can more effectively curb tumor growth, metastasis, and recurrence, thereby augmenting the overall therapeutic outcomes in cancer treatment [ 42 ]. Here, we investigated the antigen-cross-presentation of BMDCs by staining the immunodominant ovalbumin-derived SINFEKL peptide bound to MHC I. As shown in Fig. 3 E, mOVA/siPD-L1 treatment group showed a slightly increasement of MHC I complex on BMDCs surface. LNP/mOVA/siPD-L1 nanoparticles exhibited significant improvement of antigen cross-presentation, indicating the efficient expression and processing of tumor antigen on BMDCs.
The secretion of cytokines serves as another indicator of BMDCs activation. By assessing the concentration and activity of cytokines, we can gauge the degree of BMDCs activation and the status of immune responses. Concurrent with evaluating BMDCs maturation, we collected cell culture supernatants and measured the concentrations of cytokines, including IL-6 ( Fig. S4 ), IL-12p70 ( Fig. 3 F), TNF-α ( Fig. 3 G), and IFN-β ( Fig. 3 H), within these supernatants. The results indicated that, compared to the control group, the LNP/mOVA/siPD-L1 treatment group exhibited significantly increased secretion of these cytokines, reaching levels comparable to those in the LPS-positive control group. Next, we investigated the activating effect of dual-bioengineered DC vaccines on CD8 + T cells. Initially, CD8 + T cells were extracted and sorted from the spleens of OT-1 transgenic mice, and then mixed with the dual-bioengineered DC vaccines. After three days of co-culturing, we observed a substantial proliferation of CD8 + T cells, as evidenced by the dilution of the dye, in comparison to the control groups ( Fig. 3 I). Thus, our findings demonstrate that the dual-bioengineered DC vaccines serve as efficient antigen presenters and are capable of robustly stimulating T cell proliferation, thereby activating specific anti-tumor immune responses. These findings concerning BMDC activation, antigen cross-presentation, cytokine secretion, and T cell proliferation suggest that LNP/mOVA/siPD-L1-dual-bioengineered BMDCs serve as effective tumor antigen presenters with the ability to potently stimulate anti-tumor cellular immune responses.
LNP/mOVA/siPD-L1 dual-bioengineered BMDCs for prophylactic tumor therapy
To investigate the antitumor efficacy of dual-bioengineered DC vaccines, we selected OVA as the tumor-specific antigen and utilized a gene therapy strategy to transfect BMDCs, achieving efficient intracellular expression of OVA protein for antigen presentation. Additionally, siPD-L1 was used to downregulate the expression of PD-L1 protein on the surface of BMDCs, thereby enhancing their immune-activating capacity. Besides, we employed a lentiviral transfection strategy to establish CT26-OVA cells that stably express OVA protein, thereby utilizing OVA as the tumor-specific antigen for CT26 cells. LNP/mOVA/siPD-L1-dual-bioengineered BMDCs were initially explored as vaccines in a prophylactic tumor model. As depicted in Fig. 4 A and B, BALB/c mice were immunized three times via subcutaneous injection with LNP/mOVA/siPD-L1-dual-bioengineered BMDCs. Subsequently, the mice were subcutaneously inoculated with CT26-OVA cells, and tumor volume, mouse body weight, and survival curves were monitored throughout the therapeutic period.
As shown in Fig. 4 C–F, in the PBS and naked BMDCs control groups, tumors appeared at day 30 and sharply grew during the next observation period. While the tumors in dual-bioengineered BMDCs first appeared 4 days later and grew smoothly. The tumor volumes in the control groups were significantly larger than the treated groups. Compared with PBS control group, naive BMDCs group also showed a certain tumor inhibition effect, which may be due to a large number of BMDCs cell injection, which activated the anti-tumor immune response of the body to a certain extent ( Fig. 4 D). The mice in dual-bioengineered BMDCs treatment group exhibited a remarkable inhibitory effect on tumor growth, with two mice remaining tumor-free by day 19 (2/5), and the tumor volumes in the other three mice being less than 200 mm 3 ( Fig. 4 E). During the course of treatment, there was no significant reduction in body weight observed in all mice, and no notable differences in body weight were detected among mice across various treatment groups ( Fig. 4 G). This, to a certain extent, attests to the safety of the treatment strategy. The survival curves of tumor - bearing mice were analyzed to evaluate the therapeutic efficacy of different treatments ( Fig. 4 H). Mice treated with PBS showed rapid mortality, with all succumbing within 21 days. Naive DCs - treated mice had a slightly prolonged survival, but still all died by 23 days. In stark contrast, mice treated with engineered DCs exhibited significantly prolonged survival, with a survival rate of 60 % (3/5) observed on the 47th day (***p < 0.001 vs. PBS and Naive DCs groups). Furthermore, two mice in this group remained tumor-free throughout the observation period. These findings underscore the potential of dual-bioengineered BMDCs as a prophylactic tumor vaccine in effectively inhibiting tumor onset and growth. This strategy holds significant research importance and exhibits promising application prospects.
LNP/mOVA/siPD-L1 dual-bioengineered BMDCs for therapeutic tumor therapy
Compared to prophylactic cancer vaccines, therapeutic cancer vaccines offer a novel treatment approach for patients already diagnosed with cancer. By activating the patient's own immune system to target and eliminate cancer cells, these vaccines hold promise in extending survival, enhancing treatment outcomes, and providing innovative solutions to address challenges such as cancer recurrence and metastasis. Encouraged by the outcomes of the prophylactic DC vaccine treatment, we next investigated the anti-tumor therapeutic efficacy of LNP/mOVA/siPD-L1 dual-bioengineered BMDCs as a therapeutic vaccine. Firstly, CT26-OVA cells were inoculated subcutaneously into mice on day 0. On day 1, BMDCs were extracted, induced, differentiated, and transfected to obtain LNP/mRNA/siRNA-engineered BMDCs. These dual-bioengineered BMDCs were then administered as DC vaccines via subcutaneous injection on days 8, 13, and 17 ( Fig. 5 A and B). During this period, tumor size and changes in mouse body weight were monitored. On day 20, the mice were euthanized, and the content of tumor-associated immune cells in the tumor tissue, as well as the secretion of immune factors in the blood, were analyzed. As shown in Fig. 5 C, the tumor growth trend in the naive DCs treatment group was comparable to that of the PBS control group, indicating that unmanipulated DC cells struggle to achieve specific killing of tumor cells. In contrast, the dual-bioengineered DCs treatment group significantly inhibited tumor growth starting from day 13, with a tumor-free rate of 40 % (2/5) on day 19, and the tumor sizes of the three tumor-bearing mice ranged between 250 mm 3 and 550 mm 3 . The therapeutic efficacy of dual-bioengineered DCs in the therapeutic tumor model was relatively inferior to that in the prophylactic tumor model. Throughout the treatment process, there were no significant differences in body weight among the experimental groups, preliminarily demonstrating the safety of the treatment strategy ( Fig. S5 ). After the completion of anti-tumor treatment, we detected the maturation of DC cells in tumor-draining lymph nodes and the content of T EM cells in mouse spleens. In tumor-draining lymph nodes ( Fig. 5 D), the proportion of mature DC cells in dual-bioengineered DCs group was significantly higher than that in the PBS group and the naive DCs group. In the spleen, the percentage of T EM cells in dual-bioengineered DCs group was also significantly increased compared with the other two groups ( Fig. 5 E). These results indicated that dul-bioengineered DCs could effectively promote the maturation of DC cells and enhance the generation of T EM cells, thereby improving the anti-tumor immune response. Next, we analyzed the immune cell content in the tumor tissue using flow cytometry. The results showed that the levels of immune cells promoting tumor treatment, including CD4 + T cells ( Fig. 5 F), CD8 + T cells ( Fig. 5 G), and M1 macrophages ( Fig. 5 H), were significantly higher in the dual-bioengineered DCs treatment group compared to the control groups. Meanwhile, the level of Treg cells, which inhibit tumor treatment, was markedly reduced ( Fig. 5 I). This indicated that the dual-bioengineered DC vaccine played a crucial role in tumor progression. Additionally, we assessed the expression of PD-L1 protein on the surface of tumor cells ( Fig. 5 J). The results revealed that PD-L1 expression was relatively increased in dual-bioengineered DC treatment group compared to the control group, possibly due to the treatment strategy inducing a certain degree of immune tolerance.
Furthermore, we sectioned the tumor tissue, and used immunofluorescence and immunohistochemistry techniques to analyze the content of CD4 + T cells and CD8 + T cells ( Fig. 5 K, Fig. S6 ) in the tumor tissue. The immunofluorescence results were consistent with the aforementioned flow cytometry findings. After treatment with dual-bioengineered DC vaccine, while activating anti-tumor specific immunity, it also induced upregulation of PD-L1 on tumor cells ( Fig. S7 ), which may be a major reason for the less-than-ideal therapeutic effect. H&E staining of tumor tissues revealed distinct histological changes among the three groups. The PBS group showed dense tumor cell infiltration with no obvious signs of necrosis. In the naive DCs group, there was a slight reduction in tumor cell density, but no significant necrosis was observed. In contrast, the dual-bioengineered DCs group exhibited extensive necrosis and a significant reduction in viable tumor cells, indicating a stronger anti-tumor effect. This may be attributed to the enhanced ability of dual-bioengineered DCs to activate the immune system and induce tumor cell apoptosis. Ki67 staining of tumor tissues revealed that the PBS group exhibited the highest density of Ki67-positive cells, indicating strong tumor cell proliferation. Naive DCs treatment led to a moderate reduction in Ki67-positive cells compared to PBS. Engineered DCs showed the most significant decrease in Ki67-positive cells, suggesting a stronger inhibitory effect on tumor cell proliferation. These findings support the potential of engineered DCs as a promising approach for tumor therapy.
In addition, we employed the ELISA method to detect anti-tumor-related cytokines in the serum of mice after treatment. The results indicated that the levels of IL-6, IFN-γ, and TNF-α in the serum of mice from the dual-bioengineered DC vaccine group were significantly increased post-treatment, further confirming the activation of anti-tumor immunity elicited by the dual-bioengineered DC vaccine ( Fig. S8 ).These results suggest that the timing and method of administering the therapeutic DC vaccine in anti-tumor applications need further optimization to avoid inducing tumor immune suppression and thereby promoting the risk of tumor progression.
The biological safety in the research process of tumor vaccines is of paramount importance. It ensures that the vaccines do not cause unintentional harm or pose potential risks to the body during various stages such as research and development, production, and clinical application. It is a critical factor in safeguarding the safety of cancer patients and advancing the progress of tumor immunotherapy. In the current study, we evaluated the effects of the treatment on the blood biochemistry ( Fig. S9 ), hepatorenal function indicators ( Fig. S10 ), and renal function indexes ( Fig. S11 ) of mice. The results demonstrated that all examined blood biochemistry parameters, as well as the specific indicators reflecting liver and kidney function, were within the normal range after the treatment. This finding is of great significance as it indicates that the treatment did not adversely affect the general metabolic status or the specific functions of the liver and kidneys in the mice. The normal blood biochemistry results suggest that the treatment did not disrupt the balance of essential metabolites, electrolytes, or enzymes in the blood, which are crucial for maintaining normal physiological functions. Furthermore, we conducted histological examinations using HE staining to assess the potential impact of the treatment on various organs of mice. The HE staining results revealed that all examined organs, including heart, liver, spleen, lung, and kidney, exhibited normal histological structures and morphologies after the treatment ( Fig. S12 ). This finding is significant as it indicates that the treatment did not induce any detectable histological abnormalities or pathological changes in the mice's organs. The normal appearance of the tissue architecture, cell morphology, and staining patterns suggests that the treatment was well-tolerated and did not cause any adverse effects at the histological level. These results demonstrated the safety and tolerability of the treatment regimen, laying the foundation for further preclinical and clinical studies to evaluate the efficacy and safety of the treatment in a broader context.
Combination of DC vaccines with anti-PD-L1 antibody for therapeutic tumor therapy
In previous anti-tumor studies, we found that dual-bioengineered DC vaccines could effectively inhibit tumor growth in therapeutic tumor models. However, during treatment, the expression of PD-L1 on the surface of tumor cells increased, indicating that this therapy could induce a certain degree of immune tolerance. Inspired by this, we further introduced a combination therapy of dual-bioengineered DC vaccines and anti-PD-L1 antibodies to achieve synergistic anti-tumor effects. In this study, we first established a therapeutic tumor model by inoculating CT26-OVA tumor cells on day 0. DC vaccines were administered subcutaneously on days 9, 13, and 17, while anti-PD-L1 antibodies were administered via intraperitoneal injection. We monitored tumor size and mouse weight, and on day 45, naive mice and dual-bioengineered DC treated mice were inoculated with a second round of CT26-OVA tumor cells to further investigate the anti-tumor immune memory effect ( Fig. 6 A and B). The tumor inhibition results ( Fig. 6 C–G) showed that tumors in the PBS control group grew rapidly, with tumor volumes exceeding 1500 mm 3 in mice starting from day 19 ( Fig. 6 C). According to animal ethics guidelines, mice are considered dead when tumor size exceeds 1500 mm 3 . Compared to the PBS group, the anti-PD-L1 antibody treatment group inhibited tumor growth to some extent, with a tumor inhibition rate exceeding 40 % on day 21 ( Fig. 6 D). The dual-bioengineered DC treatment group showed better tumor inhibition, with an inhibition rate of 80 % on day 21 ( Fig. 6 E). The combination therapy group of dual-bioengineered DC vaccines and anti-PD-L1 antibodies achieved the best tumor inhibition. After the initial combined treatment, tumor growth was slow, and tumors gradually shrank after the third administration, becoming invisible by day 21 ( Fig. 6 F). The survival curve of mice showed distinct differences among groups. The PBS group had the earliest and most rapid decline in survival rate, with all mice not surviving beyond 25 days. The Anti-PD-L1 group had a slightly prolonged survival but still showed a marked drop, with survival ending around 25 days. The Engineered DCs group had a more favorable survival trend, yet survival rate decreased notably after 30 days. In contrast, the Engineered DCs + Anti-PD-L1 group maintained 100 % survival throughout the observation period (up to 45 days), and statistical analysis confirmed highly significant survival differences compared to other groups ( Fig. 6 H). Throughout the treatment process, there were no significant differences in mouse weight among the treatment groups ( Fig. S13 ), preliminarily indicating the safety of the treatments. After the second round of CT26-OVA tumor cells inoculation, no growth or recurrence was observed in mice from the combination therapy group ( Fig. 6 I), primarily due to the ability of anti-PD-L1 antibodies to alter the immune tolerance of tumor cells and enhance the body's specific anti-tumor killing effect.
The combination therapy of dual-bioengineered DC vaccine and anti-PD-L1 antibody for tumor treatment holds great promise. It enhances immune response against tumors by activating immune cells through vaccination and relieving immune suppression via anti-PD-L1 blockade. This approach improves treatment efficacy, broadens therapeutic options for refractory tumors. It is expected to generally enhance patients' quality of life with milder side effects, and promote personalized cancer treatment and accelerates new drug development.
Conclusions
In summary, we successfully utilized microfluidic technology to prepare lipid nanoparticles co-loaded with OVA mRNA, capable of expressing the model antigen OVA, and siRNA targeting PD-L1 for the transfection of BMDCs. This approach allowed us to construct mRNA/siRNA dual-bioengineered BMDCs, which served as tumor DC vaccines for anti-cancer research. Initially, we evaluated the physicochemical properties, cellular uptake, and transfection efficiency of the LNP/mOVA/siPD-L1 nanoparticles, confirming its stability and efficacy. Subsequently, we conducted investigations into both prophylactic and therapeutic tumor vaccine strategies. The results demonstrated that the dual-bioengineered DC vaccines effectively inhibited tumor growth. However, during the treatment process, an increase in PD-L1 protein expression on the surface of tumor cells was observed. To address this, we combined the vaccines with anti-PD-L1 antibody therapy for synergistic tumor treatment. Remarkably, this combined therapy achieved complete tumor suppression. Furthermore, rechallenge experiments provided additional evidence of the tumor immune memory effect induced by the combined therapy.
Collectively, our findings suggest that this combined therapy holds potential clinical application value in the treatment, metastasis prevention, and recurrence management of tumors. The synergistic approach not only leverages the power of dual-bioengineered DCs to stimulate an immune response against cancer but also addresses the blockade of PD1/PD-L1 pathway by synergistic anti-PD-L1 antibody therapy, enhancing the overall therapeutic efficacy. Future studies will focus on further refining this strategy and exploring its potential in clinical settings, ultimately aiming to improve patient outcomes in the fight against cancer.
CRediT authorship contribution statement
Dong Yang: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis. Jing Zhan: Visualization, Investigation, Formal analysis, Conceptualization. Ling Miao: Methodology, Investigation, Formal analysis. Chuan He: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Hong Xu: Writing – review & editing, Supervision, Project administration, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 81902966).
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2025.102357 .
Contributor Information
Chuan He, Email: chuanhe001@jlu.edu.cn.
Hong Xu, Email: x_hong@jlu.edu.cn.
Supplementary data
The following is/are the supplementary data to this article.
Multimedia component 1
Data availability
Data will be made available on request.
References
1. Gong N., Sheppard N.C., Billingsley M.M., June C.H., Mitchell M.J. Nanomaterials for T-cell cancer immunotherapy. Nat. Nanotechnol. 2021;16(1):25–36. doi: 10.1038/s41565-020-00822-y.
2. Kraehenbuehl L., Weng C.H., Eghbali S., Wolchok J.D., Merghoub T. Enhancing immunotherapy in cancer by targeting emerging immunomodulatory pathways. Nat. Rev. Clin. Oncol. 2022;19(1):37–50. doi: 10.1038/s41571-021-00552-7.
3. Matsuo K., Yoshie O., Kitahata K., Kamei M., Hara Y., Nakayama T. Recent progress in dendritic cell-based cancer immunotherapy. Cancers (Basel) 2021;13(10):2495. doi: 10.3390/cancers13102495.
4. Najafi S., Mortezaee K. Advances in dendritic cell vaccination therapy of cancer. Biomed. Pharmacother. 2023;164 doi: 10.1016/j.biopha.2023.114954.
5. Hato L., Vizcay A., Eguren I., Pérez-Gracia J.L., Rodríguez J., Pérez-Larraya J.G., Sarobe P., Inogés S., de Cerio A.L.D., Santisteban M. Dendritic cells in cancer immunology and immunotherapy. Cancers. 2024;16(5):981. doi: 10.3390/cancers16050981.
6. Wculek S.K., Cueto F.J., Mujal A.M., Melero I., Krummel M.F., Sancho D. Dendritic cells in cancer immunology and immunotherapy. Nat. Rev. Immunol. 2020;20(1):7–24. doi: 10.1038/s41577-019-0210-z.
7. Laureano R.S., Sprooten J., Vanmeerbeerk I., Borras D.M., Govaerts J., Naulaerts S., Berneman Z.N., Beuselinck B., Bol K.F., Borst J., Coosemans A., Datsi A., Fucikova J., Kinget L., Neyns B., Schreibelt G., Smits E., Sorg R.V., Spisek R., Thielemans K., Tuyaerts S., De Vleeschouwer S., de Vries I.J.M., Xiao Y., Garg A.D. Trial watch: dendritic cell (DC)-based immunotherapy for cancer. OncoImmunology. 2022;11(1) doi: 10.1080/2162402X.2022.2096363.
8. Cheever M.A., Higano C.S. PROVENGE (Sipuleucel-T) in prostate cancer: the first FDA-approved therapeutic cancer vaccine. Clin. Cancer Res. 2011;17(11):3520–3526. doi: 10.1158/1078-0432.CCR-10-3126.
9. Harari A., Graciotti M., Bassani-Sternberg M., Kandalaft L.E. Antitumour dendritic cell vaccination in a priming and boosting approach. Nat. Rev. Drug Discov. 2020;19(9):635–652. doi: 10.1038/s41573-020-0074-8.
10. Das R., Ge X., Fei F., Parvanian S., Weissleder R., Garris C.S. Lipid Nanoparticle-mRNA engineered dendritic cell based adoptive cell therapy enhances cancer immune response. Small Methods. 2025;9(1) doi: 10.1002/smtd.202400633.
11. Chen M.Y., Zhang F., Goedegebuure S.P., Gillanders W.E. Dendritic cell subsets and implications for cancer immunotherapy. Front. Immunol. 2024;15 doi: 10.3389/fimmu.2024.1393451.
12. Lei J., Qi S., Yu X., Gao X., Yang K., Zhang X., Cheng M., Bai B., Feng Y., Lu M., Wang Y., Li H., Yu G. Development of mannosylated lipid nanoparticles for mRNA cancer vaccine with high antigen presentation efficiency and immunomodulatory capability. Angew Chem. Int. Ed. Engl. 2024;63(13) doi: 10.1002/anie.202318515.
13. Bitounis D., Jacquinet E., Rogers M.A., Amiji M.M. Strategies to reduce the risks of mRNA drug and vaccine toxicity. Nat. Rev. Drug Discov. 2024;23(4):281–300. doi: 10.1038/s41573-023-00859-3.
14. Guo J., De May H., Franco S., Noureddine A., Tang L., Brinker C.J., Kusewitt D.F., Adams S.F., Serda R.E. Cancer vaccines from cryogenically silicified tumour cells functionalized with pathogen-associated molecular patterns. Nat. Biomed. Eng. 2022;6(1):19–31. doi: 10.1038/s41551-021-00795-w.
15. Shuwari N., Inoue C., Ishigami I., Jingushi K., Kamiya M., Kawakami S., Tsujikawa K., Tachibana M., Mizuguchi H., Sakurai F. Small extracellular vesicles carrying reovirus, tumor antigens, interferon-beta, and damage-associated molecular patterns for efficient tumor treatment. J Control Release. 2024;374:89–102. doi: 10.1016/j.jconrel.2024.07.079.
16. Chen Y., Zhang Y., Wang B., Fan Q., Yang Q., Xu J., Dai H., Xu F., Wang C. Blood clot scaffold loaded with liposome vaccine and siRNAs targeting PD-L1 and TIM-3 for effective DC activation and cancer immunotherapy. ACS Nano. 2023;17(1):760–774. doi: 10.1021/acsnano.2c10797.
17. Liu L.C., Chen J.H., Bae J., Li H.Y., Sun Z.C., Moore C., Hsu E., Han C.H., Qiao J., Fu Y.X. Rejuvenation of tumour-specific T cells through bispecific antibodies targeting PD-L1 on dendritic cells. Nat. Biomed. Eng. 2021;5(11):1261–1273. doi: 10.1038/s41551-021-00800-2.
18. Galassi C., Chan T.A., Vitale I., Galluzzi L. The hallmarks of cancer immune evasion. Cancer Cell. 2024;42(11):1825–1863. doi: 10.1016/j.ccell.2024.09.010.
19. Zhou J.S., Li Y.Y., Jiang X.H., Xin Z.Y., Liu W.S., Zhang X.Y., Zhai Y.H., Zhang Z.Z., Shi T., Xue M.H., Zhang M.Y., Wu Y., Chu Y.H., Wang S.M., Jin X., Zhu W.P., Gao J. PD-L1 siRNA incorporation into a cationic liposomal tumor mRNA vaccine enhances cytotoxic T cell activation and prevents immune evasion. Mater. Today Bio. 2025;31 doi: 10.1016/j.mtbio.2025.101603.
20. Liu J., Chen Z., Li Y., Zhao W., Wu J., Zhang Z. PD-1/PD-L1 checkpoint inhibitors in tumor immunotherapy. Front. Pharmacol. 2021;12 doi: 10.3389/fphar.2021.731798.
21. Liu S., Wu J., Feng Y., Guo X., Li T., Meng M., Chen J., Chen D., Tian H. CD47KO/CRT dual-bioengineered cell membrane-coated nanovaccine combined with anti-PD-L1 antibody for boosting tumor immunotherapy. Bioact. Mater. 2023;22:211–224. doi: 10.1016/j.bioactmat.2022.09.017.
22. Wang K., Chen J., Lin L., Yan N., Yang W.H., Cai K.Y., Tian H.Y., Chen X.S. Anion receptor-mediated multicomponent synergistic self-assembly of porphyrin for efficient phototherapy to elicit tumor immunotherapy. Nano Today. 2022;46
23. Wu J.Y., Feng Y.J., Guo X.Y., Meng M., Li H.X., Fang H.P., Li Z., Lin L., Guo Z.P., Chen J., Tian H.Y., Chen X.S. A Versatile nanovaccine enhancement strategy based on suction-inspired physical therapy. ACS Nano. 2024;18(6):4957–4971. doi: 10.1021/acsnano.3c10623.
24. Ghosh C., Luong G., Sun Y. A snapshot of the PD-1/PD-L1 pathway. J. Cancer. 2021;12(9):2735–2746. doi: 10.7150/jca.57334.
25. Meng M., Wu J., Feng Y., Lin L., Chen J., Pang X., Li Y., Hao K., Tian H., Chen X. A comprehensive strategy based on high clinical translational nanosystem for programmable immunotherapy of triple negative breast cancer. Adv Mater. 2024;36(27) doi: 10.1002/adma.202314309.
26. Yi M., Zheng X., Niu M., Zhu S., Ge H., Wu K. Combination strategies with PD-1/PD-L1 blockade: current advances and future directions. Mol. Cancer. 2022;21(1):28. doi: 10.1186/s12943-021-01489-2.
27. Kang D.D., Hou X., Wang L., Xue Y., Li H., Zhong Y., Wang S., Deng B., McComb D.W., Dong Y. Engineering LNPs with polysarcosine lipids for mRNA delivery. Bioact. Mater. 2024;37:86–93. doi: 10.1016/j.bioactmat.2024.03.017.
28. Nelson A.L., Mancino C., Gao X., Choe J.A., Chubb L., Williams K., Czachor M., Marcucio R., Taraballi F., Cooke J.P., Huard J., Bahney C., Ehrhart N. beta-catenin mRNA encapsulated in SM-102 lipid nanoparticles enhances bone formation in a murine tibia fracture repair model. Bioact. Mater. 2024;39:273–286. doi: 10.1016/j.bioactmat.2024.05.020.
29. Fan Y.C., Rigas D., Kim L.J., Chang F.P., Zang N.Z., Mckee K., Kemball C.C., Yu Z.X., Winkler P., Su W.C., Jessen P., Hura G.L., Chen T., Koenig S.G., Nagapudi K., Leung D., Yen C.W. Physicochemical and structural insights into lyophilized mRNA-LNP from lyoprotectant and buffer screenings. J. Contr. Release. 2024;373:727–737. doi: 10.1016/j.jconrel.2024.07.052.
30. Guo X.Y., Yang Z.Y., Guo Z.P., Lai H.Y., Meng H.Y., Meng M., Li T., Li Z., Chen J., Feng Y.J., Pang X., Tian H.Y., Chen X.S. A polymeric mRNA vaccine featuring enhanced site-specific mRNA delivery and inherent STING-stimulating performance for tumor immunotherapy. Adv. Mater. 2025;37(17) doi: 10.1002/adma.202410998.
31. Zhang Z.J., Meng Y.M., Guo Y.X., He X., Liu Q.R., Wang X.F., Shan F.P. Rehmannia glutinosa polysaccharide induces maturation of murine bone marrow derived dendritic cells (BMDCs) Int. J. Biol. Macromol. 2013;54:136–143. doi: 10.1016/j.ijbiomac.2012.12.005.
32. Chen J., Fang H., Hu Y., Wu J., Zhang S., Feng Y., Lin L., Tian H., Chen X. Combining mannose receptor mediated nanovaccines and gene regulated PD-L1 blockade for boosting cancer immunotherapy. Bioact. Mater. 2022;7:167–180. doi: 10.1016/j.bioactmat.2021.05.036.
33. Yu H.W., Wen Y.F., Yu W.Q., Lu L., Yang Y., Liu C.Y., Hu Z.X., Fang Z.T., Huang S.L. Optimized circular RNA vaccines for superior cancer immunotherapy. Theranostics. 2025;15(4):1420–1438. doi: 10.7150/thno.104698.
34. Jin C.Y., Zhang Y., Li B.F., Gao T.C., Wang B., Hua P.Y. Robust anti-tumor immunity through the integration of targeted lipid nanoparticle-based mRNA nanovaccines with PD-1/PD-L1 blockade. Mater. Today Bio. 2024;27 doi: 10.1016/j.mtbio.2024.101136.
35. Nie X.X., Shi C.H., Chen X.W., Yu C.C., Jiang Z.Y., Xu G.X., Lin Y., Tang M.T., Luan Y.X. A single-shot prophylactic tumor vaccine enabled by an injectable biomembrane hydrogel. Acta Biomater. 2023;169:306–316. doi: 10.1016/j.actbio.2023.08.010.
36. Pundavela J., Dinglasan S.A., Touvron M., Hummel S.A., Hu L., Rizvi T.A., Choi K., Hildeman D.A., Ratner N. Stimulator of interferon gene facilitates recruitment of effector CD8 T cells that drive neurofibromatosis type 1 nerve tumor initiation and maintenance. Sci. Adv. 2024;10(42) doi: 10.1126/sciadv.ado6342.
37. Liu H.Y., Zhou C., Jiang H.H., Chu T.Q., Zhong R.B., Zhang X.Y., Shen Y.C., Han B.H. Prognostic role of serum cytokines level in non-small cell lung cancer patients with anti-PD-1 and chemotherapy combined treatment. Front. Immunol. 2024;15 doi: 10.3389/fimmu.2024.1430301.
38. Chang H., Wen X.Y., Li Z.M., Ling Z.X., Zheng Y.T., Xu C.J. Co-delivery of dendritic cell vaccine and anti-PD-1 antibody with cryomicroneedles for combinational immunotherapy. Bioeng Transl Med. 2023;8(5) doi: 10.1002/btm2.10457.
39. Yu J.F., Sun H., Cao W.J., Song Y.P., Jiang Z.X. Research progress on dendritic cell vaccines in cancer immunotherapy. Exp. Hematol. Oncol. 2022;11(1):3. doi: 10.1186/s40164-022-00257-2.
40. Filin I.Y., Kitaeva K.V., Rutland C.S., Rizvanov A.A., Solovyeva V.V. Recent advances in experimental dendritic cell vaccines for cancer. Front. Oncol. 2021;11 doi: 10.3389/fonc.2021.730824.
41. Huang P., Jiang L.S., Pan H., Ding L.W., Zhou B., Zhao M.Y., Zou J.H., Li B.H., Qi M.W., Deng H.Z., Zhou Y.F., Chen X.Y. An integrated polymeric mRNA vaccine without inflammation side effects for cellular immunity mediated cancer therapy. Adv. Mater. 2023;35(3) doi: 10.1002/adma.202207471.
42. Miao L., Zhang Y., Huang L. mRNA vaccine for cancer immunotherapy. Mol. Cancer. 2021;20(1):41. doi: 10.1186/s12943-021-01335-5.
43. Min Y., Roche K.C., Tian S., Eblan M.J., McKinnon K.P., Caster J.M., Chai S., Herring L.E., Zhang L., Zhang T., DeSimone J.M., Tepper J.E., Vincent B.G., Serody J.S., Wang A.Z. Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy. Nat. Nanotechnol. 2017;12(9):877–882. doi: 10.1038/nnano.2017.113.
Associated Data
Supplementary Materials
Multimedia component 1
Data Availability Statement
Data will be made available on request.