Cd73-LNPs promotes antitumor T-cell immunity via amplifying Tlr3-mediated immunostimulatory dendritic cell activation

Cd73-LNPs promotes antitumor T-cell immunity via amplifying Tlr3-mediated immunostimulatory dendritic cell activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cd73-LNPs promotes antitumor T-cell immunity via amplifying Tlr3-mediated immunostimulatory dendritic cell activation Yuandong Xu, Fei Cao, Haowei Qiu, Yi Zhang, Yiting Wang, Zhen Xu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8715323/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: mRNA vaccines have emerged as highly promising therapeutic agents in the field of cancer immunotherapy. Nevertheless, the current limited immunogenicity of target antigens, along with a lack of systemic immune response, poses significant challenges to the effective implementation of mRNA vaccines. This study sought to evaluate the immunogenicity of the Cd73 protein and elucidate the mechanisms through which the Cd73 mRNA vaccines mediated its antitumor immune effects in murine models. Methods: In vitro transcription synthesis of Cd73 mRNA was employed to prepare a Cd73 mRNA vaccine based on lipid nanoparticles (Cd73-LNPs) using a microfluidic device. The immunogenicity of Cd73 was assessed via ELISPOT assays. The capability of Cd73-LNPs to activate anti-tumor immune responses was investigated via flow cytometry. The therapeutic efficacy of Cd73-LNPs was tested in models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Furthermore, the mechanism of action of Cd73-LNPs was elucidated through single-cell transcriptomics sequencing and RNA transcriptomics sequencing. Results: The Cd73 protein exhibited potent immunogenicity. In vitro, Cd73-LNPs have been observed to promote the maturation and activation of primary dendritic cells. In vivo, they have been observed to activate both humoral and cellular immune responses, stimulating secretion of Ifn-γ and granzyme B, effectively suppressing tumor growth in models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Mechanistically, Cd73-LNPs activate dendritic cells through toll-like receptor 3 signaling, enhancing their activation and upregulating chemokine receptor expression on Cd8 + T cells, which promotes Cd8 + T cell infiltration into the tumor microenvironment. Safety evaluations revealed that Cd73-LNPs do not produce toxic side effects on vital organs. Conclusion: Cd73 mRNA vaccines have been demonstrated to safely and effectively induce antitumor immunity through toll-like receptor 3 signaling, indicating considerable potential for clinical application. Cancer mRNA vaccines Cd73 Tlr3 Cd8+ T cells Immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background mRNA vaccines have emerged as a promising platform for cancer immunotherapy, characterized by high potency, safe administration, rapid development potential, and cost-effective manufacturing[1]. These vaccines function by encoding tumor-specific antigens or costimulatory molecules, which are subsequently translated into proteins by the patient's cells, particularly antigen-presenting cells, thereby eliciting a robust anti-tumor immune response [2–4]. Despite significant advancements in the field of cancer mRNA vaccines, challenges persist, particularly concerning the poor immunogenicity of antigenic targets and the inability to induce rapid yet adaptable immune responses, which are likely necessary to suppress rapidly evolving cancers[5]. Identifying immunogenic antigenic targets holds substantial potential for accelerating the clinical implementation of mRNA vaccines. CD73, a membrane-bound ecto-5'-nucleotidase, facilitates the extracellular conversion of adenosine monophosphate into the immunosuppressive molecule adenosine[6]. Its upregulation is frequently observed across various tumor types, including melanoma, ovarian cancer, and prostate cancer, thereby contributing to the accumulation of adenosine within the tumor microenvironment and promoting immune evasion[7]. In addition to its enzymatic activity, CD73 plays a critical role in cell adhesion and migration in cancers[8]. Moreover, CD73 expression has been detected on a range of immune cells, such as T cells, macrophages, and natural killer cells, and its involvement in the immunosuppressive functions of these cells has been elucidated[9–11]. Given the evidence of CD73's immunosuppressive and pro-tumor effects, the targeted inhibition of the CD73 pathway has emerged as a highly promising therapeutic strategy. Significant efforts have been devoted to the development of CD73-targeting therapeutics, such as small-molecule inhibitors, monoclonal antibodies, and bispecific antibodies[6, 12]. Nonetheless, no such drug has yet advanced to the clinical stage. Our previous research developed lipid nanoparticle (LNP) delivery systems specifically targeting the spleen and draining lymph nodes [13]. In the current study, building upon our prior work with LNPs, we examined the therapeutic efficacy and underlying mechanisms of Cd73 mRNA vaccines (Cd73-LNPs) in a mouse model. The findings demonstrated that Cd73-LNPs elicit robust antitumor immune responses in vivo through antigen presentation pathways, facilitating the secretion of effector molecules such as Ifn-γ and granzyme B (Gra B) by Cd8 + T cells, and effectively inhibiting tumor growth in models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Mechanistically, Cd73-LNPs were shown to upregulate chemokine receptor expression on Cd8 + T cell surfaces, thereby enhancing Cd8 + T cell infiltration into the tumor microenvironment. Additionally, our research indicated that Cd73-LNPs activate dendritic cells by stimulating the Toll-like receptor 3 (Tlr3) signaling pathway, thereby promoting dendritic cell activation. This study underscores the strong immunogenicity of the Cd73 protein and highlights the significant potential of Cd73 mRNA vaccines for clinical translation. Methods Culture of cell lines The B16-OVA, TC-1, ID8, and RM-1 cells were cultivated in RPMI-1640 medium (Gibco, USA) with the addition of 10% foetal calf serum (Gibco, USA) and 1% Penicillin-Streptomycin Solution (Pricella, USA). All cell lines were obtained from the American Type Culture Collection (ATCC) website. A regular programme of checks was implemented for the purpose of verifying the absence of mycoplasma contamination in all cell lines. The methodology employed for this purpose was the MycAwayTM Plus-Color One-Step Mycoplasma Detection Kit (YEASEN, China). The isolation of bone marrow-derived dendritic cells (BMDCs) was achieved by harvesting bone marrow from the femurs and tibias of C57BL/6J mice. Following red blood cell lysis using ACK lysis buffer, the remaining bone marrow cells were cultured in RPMI 1640 medium, which was supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, 20 ng/mL recombinant GM-CSF (rm-GM-CSF, Peprotech, USA) and 20 ng/mL recombinant IL-4 (rm-IL-4, Peprotech, USA). This was done to promote dendritic cell differentiation. The medium was replaced at two-day intervals, with half of it being replaced in order to maintain optimal growth conditions. On the sixth day, the immature BMDCs were harvested from the culture dish and utilised in subsequent experiments. mRNA synthesis The transcription of Cd73 mRNA, E7 mRNA, Psma mRNA and Luciferase mRNA was conducted in vitro, utilising T7 RNA polymerase (Megascript, Ambion, USA) and linearized plasmid templates. In the transcription reaction, methylated pseudouridine (m1Ψ)-5′-triphosphate (TriLink) was utilised as a substitute for the conventional uridine (UTP), thereby introducing modifications to the mRNA nucleotides. Subsequently, the synthesised mRNA was 5′-capped using 2′-O-methyltransferase (ScriptCap; CellScript, USA) and an m7G capping kit to enhance its stability and translation efficiency. The synthesis of mRNA was followed by a purification process, which involved the use of a cellulose column (Sigma-Aldrich, USA), with the aim of ensuring the removal of any impurities. The synthesis of mRNA was subjected to rigorous quality control measures. This was achieved by means of capillary electrophoresis, utilising the Agilent Technologies 5200 Fragment Analyzer (Agilent Technologies, USA). The integrity of the mRNA was assessed using the DNF-472-1000 kit and gel retardation method. The synthetic mRNA samples were stored at -80°C until further use. mRNA-LNPs Preparation and characterization The ionizable amino lipid (E12A1A3) synthesis and complete methodology for mRNA-LNPs synthesis have been thoroughly described in our previous study[13]. The organic phase dissolved in ethanol (the ethanol phase) at a molar ratio of E12A1A3/DSPC (Avanti. China)/cholesterol (Avanti. China)/DMG-PEG 2000 (Avanti. China) of 50:10:38.5:1.5. Meanwhile, the mRNA was dissolved in sodium citrate solution (the aqueous phase). The ethanol and aqueous phases were combined at a volume ratio of 1:3, with a lipid-to-mRNA N/P ratio of 5:1. The subsequent step involved the amalgamation of lipids and mRNA, which was achieved by means of a syringe pump within a microfluidic chip device (INanoE, MicroNano, China). The final product was then subjected to a centrifugal process at 1200 × g for 10 minutes, utilising a 100 kDA ultrafiltration membrane. This was followed by a thorough washing procedure and resuspension in Phosphate Buffered Saline (PBS). The preparations were stored at a temperature of 4°C. The particle size distribution of the mRNA-LNPs was analysed using dynamic light scattering (DLS) on a Zetasizer Nano (Malvern Instruments, Malvern, UK). The Quant-iT RiboGreen RNA Assay Kit (Invitrogen, USA) was utilised to evaluate concentration and encapsulation efficiency. mRNA-LNPs cytotoxicity test in vitro and in vivo The BMDCs were then exposed to different concentrations of mRNA-LNPs or PBS for 24 hours. After this, the BMDCs were collected and the number of living cells was measured using a kit called the Cell Counting Kit-8 (CCK8) (Beyotime, China). In short, the cells were put in a 96-well plate at a density of 1×10 4 –1×10 5 cells/well and left to settle for overnight. After 24 hours, the cells were treated with different concentrations of mRNA-LNPs or PBS as a control. After treatment, 10 µL of CCK8 solution was added to each well and the plate was left for two more hours at 37°C. The amount of light absorbed at 450 nm was measured using a special machine, and the number of living cells was calculated as a percentage of the control group. We used this formula to work out how many cells were alive: Relative viability (%) = [(OD _treated − OD _blank) / (OD _control − OD _blank )] × 100. Serum samples were collected from tumor-free mice following Cd73-LNPs administration for biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and Creatine Kinase (CK), undertake a comprehensive evaluation of hepatic and renal function. Real-Time PCR The TRIzol Reagent (Invitrogen) was utilised for the isolation of total cellular RNA, in accordance with the manufacturer's instructions. The synthesis of cDNA was accomplished through the employment of the Hifair II 1st Strand cDNA Synthesis Kit (Yeasen). The quantification of target mRNA expression was performed using the Hieff UNICON Universal Blue qPCR SYBR Green Master Mix Kit (Yeasen). Subsequent analysis of the results was conducted within the Bio-Rad detection system. The relative target mRNA expression was calculated by using a 2ˆ-ΔΔCT method and normalized to the expression of 𝛽-actin. The primer sequences utilised in the present study as showed in previous article and Table 2[13]. Enzyme-Linked Immunosorbent Assays (ELISA) Relative ELISA kits were procured from eBioscience with the objective of detecting the cytokines (Il-1𝛽, Il-2, Il-6, and Il-12P70) released from BMDCs culture supernatant. ELispot assay for Cd73-LNPs Enzyme-Linked Immunospot (ELISpot): IFN-𝛾 production by splenocytes isolated from post-immunized mice was measured by ELISpot. The ELISpot was performed strictly as described[13]. Bone marrow-derived dendritic cells (BMDCs) were differentiated from C57BL/6J mice and treated with Cd73-LNPs for a period of 24 hours. T cells were isolated from the spleens of C57BL/6J mice and co-cultured with mRNA-treated BMDCs at a ratio of 1:10 for a further 24 to 48 hours. Following the incubation period, IFN-γ secretion by activated T cells was quantified using an IFN-γ ELISpot assay (Dakewe, China). The wells were pre-coated with an anti-IFN-γ capture antibody, and after incubation, spot formation was visualised using a biotinylated detection antibody and streptavidin-alkaline phosphatase conjugate, eflecting its immunogenic potential. Animal experiment The Institutional Animal Care and Use Committee (IACUC) at Sun Yat-Sen University has formally approved all the animal procedures (SYSU-IACUC-2025-002827). All animal experiments were performed under pathogen-free conditions, in compliance with the Laboratory Animal Care and Use Guidelines set by the National Institute of Health. C57BL/6 mice, aged between 6 and 8 weeks, were procured from Gemmatech, Tlr3 −/− mice purchased from Cyagen. The subcutaneous tumor models employed in the present study included B16-OVA, TC-1, ID8, and RM-1-Psma, with tumor cell inoculation doses of 5x10 5 , 5x10 5 , 1x10 6 and 2.5x10 5 cells, respectively. Each treatment group comprised a minimum of five mice. Immunization (0.5 mg/kg mRNA-LNPs, intramuscular injection) was initiated upon the tumors reaching a size of 100 mm 3 or less. Tumor size was measured with calipers at 2–3 days intervals until the tumor volume reached an endpoint of 1500 mm³. The tumor volume was calculated using the following formula: The formula for calculation is as follows: length x width x width. The euthanasia of mice was conducted in accordance with the following criteria: a loss of ≥ 20% of body weight, an inability to consume food, or the presence of tumors measuring ≥ 1.5 cm³. Flow cytometry analysis BMDCs, spleens and lymph nodes were extracted from mice bearing tumors, and subsequently analyzed using flow cytometry to evaluate immune cell populations and their functional characteristics following various treatments. For further detail, please refer to the previously published article[14]. As for the blood samples, following the lysis of the blood then stained in a manner consistent with that of the spleen specimens. The staining of Ifn-γ, Tnf-α, and Gra B in tumor microenvironment Cd8 + T cells necessitates a series of processing steps, including membrane permeabilisation and fixation. Subsequent to staining, analysis of the cells was conducted via flow cytometry, thereby enabling the assessment of the frequency and activation status of discrete immune cell populations. The acquisition of data was conducted utilising a Beckman Coulter flow cytometer, and subsequent analyses were executed employing FlowJo or CytExpert software. The expression levels of surface markers were then quantified in order to evaluate the contribution of different immune cell populations to immune response. Quantification of adenosine by LC-MS/MS The tissue sample should be precisely weighed (0.1 g/0.5 g) and transferred into a 5-mL grinding tube. The tube should then be diluted to 0.5/1 mL, and the sample homogenised. Finally, 100 µL of tissue and serum should be taken. The addition of 100 µL of a 2.5% perchloric acid aqueous solution is required, followed by ultrasonication in order to precipitate proteins. The centrifuge should be operated at 12,000 rpm for a period of 20 minutes. Subsequently, 150 µL of the above mixture should be transferred and neutralised with 15 µL of a 1 mol/L NaOH aqueous solution. The mixture should be thoroughly agitated and subsequently filtered through a 0.22 µm membrane. The transfer of the sample to a brown sample vial is required for the subsequent instrumental analysis (Agilent HPLC 1200). scRNA-seq and data analysis for mouse tumors For each scRNA-seq sample, subcutaneous tumors from three mice within the same group were pooled. Tumors were generated by subcutaneously injecting 2.5 × 10⁵ RM1-Psma cells into 8-week-old C57BL/6 mice. After Cd73-LNPs treatments, the tumors collected from PBS groups and Cd73-LNPs were individually processed and combined into a single-cell suspension. Cd45 + immune cells were isolated by staining the suspension with anti-mouse Cd45 for 30 min and sorted using a BD FACSAriaIII for downstream scRNA-seq analysis. A total of 20 clusters were generated at a resolution of 0.6, and further identified into “Cd4 T”, “Cd8 T”, “NK”, “Macrophage”, “Monocyte”, “B cell”, “pDC”, “cDC1”, “cDC2”, “Neutrophil” and “unidentified” (not shown) using the expression of published markers. A detailed analysis of the steps involved, please refer to the references provided[15]. Statistical analysis All statistical analyses were conducted with GraphPad Prism (Version 8.0, CA, USA). A p value less than 0.05 was considered statistically significant (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant). Results Safety profile and in vivo immunogenicity of Cd73 protein induced by Cd73-LNPs. The preparation of Cd73 mRNA vaccines (Cd73-LNPs) utilized lipid nanoparticles previously developed in our research, as depicted schematically in Fig. 1 A. The in vitro transcription of CD73 mRNA was evaluated using capillary electrophoresis, confirming that the mRNA met quality control standards, with no signs of degradation or residual impurities ( Fig. 1 B, Figure S1 ) . We conducted a comprehensive characterization of the physical and chemical properties of the Cd73-LNPs. The average diameter of the Cd73-LNPs complex was determined to be 81.23 ± 0.58 nm, with a polydispersity index (PDI) of 0.095 ± 0.017 ( Fig. 1 C, Table 1) . Subsequently, we evaluated the translational efficiency of the Cd73-LNPs. Both western blot and flow cytometry analyses indicated that the transfection of BMDCs with Cd73-LNPs resulted in effective translation into Cd73 protein ( Fig. 1 D-F, Figure S2 ) . The cytotoxicity of Cd73-LNPs in vitro was assessed using the CCK8 assay, which revealed that administration at a concentration of 10 µg/mL did not significantly affect the viability of BMDCs ( Fig. 1 G ) . Safety evaluations were conducted following the administration of Cd73-LNPs to ascertain that the mice exhibited a favorable tolerance to the therapy. Blood biochemical analyses revealed that key indicators of liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and creatine kinase (CK), remained within normal physiological ranges, suggesting the absence of hepatic or renal dysfunction ( Fig. 1 H ) . Having established that Cd73-LNPs possess favorable characterization and safety profiles, the subsequent phase of the research involved assessing the immunogenicity of the Cd73 protein. In healthy C57BL/6 mice, following four intramuscular injections of Cd73-LNPs (5 µg per mouse), T cells from the spleen were co-cultured with BMDCs pretreated with Cd73-LNPs. Interferon-gamma (IFN-γ) production was quantified using an ELISpot assay ( Fig. 1 I ) . The results demonstrated that BMDCs pretreated with Cd73-LNPs exhibited a significantly enhanced production of Ifn-γ compared to control BMDCs. This finding indicates that Cd73-LNPs effectively activated specific immune responses, thereby underscoring their potential as tumor-associated antigen mRNA vaccines ( Fig. 1 J-K ). The findings of this study illustrate the successful synthesis and delivery of Cd73 mRNA using lipid nanoparticles (LNPs), thereby verifying their effective expression within dendritic cells. Additionally, the research substantiates both the in vitro and in vivo safety profiles of Cd73-LNPs and demonstrates their capacity to activate immune responses, highlighted the potential of Cd73-LNPs as tumor-associated antigen mRNA vaccines. Cd73-LNPs induced the maturation and activation of BMDCs. The efficient presentation of antigens by antigen-presenting cells (APCs) and the subsequent activation of adaptive immune cells are crucial for the efficacy of mRNA vaccines [16] ( Fig. 2 A ) . This study undertook a comprehensive investigation into the phenotypic modulation of BMDCs by Cd73-LNPs. Phenotypic alterations in BMDCs were assessed 24 hours post-transfection with Cd73-LNPs. Given that innate immune signaling often facilitates the expression of inflammatory cytokine genes to activate dendritic cells, we first evaluated cytokine gene expression in BMDCs using quantitative PCR (qPCR). The findings indicated that Cd73-LNPs treatment significantly induced the expression of proinflammatory cytokine genes Il-12p40, Ifn-α1, Ifn-α4, and Ifn-β1 in BMDCs, with a milder induction observed for Il-1β, Il-6, and Il-12α ( Fig. 2 B ) . ELISA results corroborated the upregulation of protein levels of Il-1β, Il-6, Il-12p70, and Ifn-β cytokines in the culture supernatant from cells treated with Cd73-LNPs (Fig. 2 C). Consequently, it was determined that Cd73-LNPs induced the expression of proinflammatory cytokines in BMDCs. Furthermore, flow cytometry analysis revealed that Cd73-LNPs significantly upregulated the surface expression of costimulatory molecules, including Cd80, Cd86, H-2m, Cd40, Cd103, Ox40l, and 4-1bbl on BMDCs, indicating maturation and activation of these cells ( Fig. 2 D-H, Figure S3) . Additionally, the expression of Ccr7 was upregulated (Figure I, Figure S3) , which is essential for the directional homing of dendritic cells to lymphoid tissues[17]. These findings suggested that Cd73-LNPs can effectively enhance the expression of cytokines and costimulatory molecules in BMDCs, highlighting their potential to activate and mature dendritic cells for enhanced immunotherapeutic efficacy. Building upon these results, future studies will investigate the functional implications of these phenotypic changes in eliciting robust anti-tumor immune responses. Cd73-LNPs activated potent humoral and cellular immunity in vivo . mRNA vaccines are recognized for their ability to induce robust immune responses by activating both humoral and cellular immunity[2]. The roles of Cd73-LNPs in the in vivo immune response were investigated based on the phenotypic modulation of BMDCs by Cd73-LNPs. In tumor-free C57BL/6 mice models, after two rounds of immunization, serum samples and T cells isolated from spleen were collected for functional assessment (Fig. 3 A). CD73 serves as the rate-limiting enzyme in adenosine metabolism, however, our findings indicated that Cd73-LNPs did not significantly alter serum adenosine levels ( Fig. 3 B ) . Notably, a decrease in serum Cd73 protein concentrations was observed, accompanied by an increase in Cd73-specific immunoglobulin G (IgG) concentrations ( Fig. 3 B ) , suggesting that Cd73-LNPs effectively elicited a humoral immune response. As the largest secondary lymphoid organ, the spleen serves as a critical site for the initiation of immune responses by mRNA vaccines[18, 19]. Our observations indicate that Cd73-LNPs significantly increased the proportion of Cd3 + T cells in the spleen, with a particular emphasis on Cd8 + T cells. In contrast, there was a notable decrease in the proportion of Cd11 + dendritic cells; however, their surface expression of Cd80 and Cd86 was elevated. Additionally, the proportion of F4/80 + /Cd206 + macrophages was diminished, suggesting a transformation of these macrophages. No significant changes were observed in the proportions of natural killer (NK) cells and B cells ( Fig. 3 C-D, Figure S4-5) . Given that Cd73-LNPs were associated with increased serum concentrations of specific IgG, we further evaluated the proportion of activated plasma cells in the spleen. The results revealed a higher proportion of B220/Cd138-positive plasma cells, indicating that Cd73-LNPs facilitate B cell activation ( Fig. 3 E, Figure S6) . Furthermore, we found that Cd73-LNPs promote the activation of Cd4 + and Cd8 + T cells in vitro (Figure S7) and enhance the secretion of effector molecules such as Ifn-γ, Granzyme B, and Tnf-α by Cd8 + T cells in vivo ( Fig. 3 F-H, Figure S8) . Moreover, the transcription factor Eomes, known for its role in the activation of Cd8 + T cells[20], was observed to have increased expression ( Fig. 3 I, Figures S8-9 ). These results indicate that Cd73-LNPs can effectively enhance both humoral and cellular immune responses in vivo without affecting serum adenosine concentrations. Cd73-LNPs improved immune cell infiltration in the tumor microenvironment and enhanced anti-tumor efficacy It has been demonstrated that Cd73-LNPs possess favorable safety profiles and can effectively activate both humoral and cellular immune responses in vivo. We next examined the anti-tumor efficacy of Cd73-LNPs in subcutaneous tumor models, including B16-Ova (melanoma), TC-1 (cervical cancer), ID8 (ovarian cancer) and RM1-Psma (prostate cancer). E7 is a specific mRNA vaccine therapeutic target for the TC-1 models[21], and prostate specific membrane antigen (PSMA) is considered an effective target for prostate cancer treatment, has been validated in CAR-T, and mRNA vaccines[22]. In this study, following administration of the four Cd73-LNPs, a significant advantage in both tumor volume reduction and overall survival in the aforementioned models ( Fig. 4 A-I, Figure S10 ). Tumor volumes consistently remained below 500 mm³ after Cd73-LNPs treatments, demonstrating superior antitumor ability of Cd73-LNPs. The complete response rates for B16-Ova model, TC-1 model and ID8 model were 40% (2/5), 60% (3/5) and 100% (5/5), respectively. In the RM1-Psma model, Cd73-LNPs demonstrated significantly superior efficacy compared to Psma-LNPs, in the TC-1 model, the efficacy of Cd73-LNPs was comparable to that of the mRNA vaccine targeting the specific antigen E7. Prostate cancer is characterized as an immunologically cold tumor, marked by a tumor microenvironment deficient in effective immune cell infiltration[23]. Surprisingly, Cd73-LNPs have been observed to enhance immune cell infiltration in the prostate cancer tumor microenvironment, including Cd45-positive immune cells, Cd8 + T cells, and NK cells (Fig. 4 J, Figure S11 ). In addition, Cd73-LNP have been shown to facilitate the secretion of Ifn-γ and Gra B by Cd8 + T cells within the tumor microenvironment in prostate cancer model (Fig. 4 K-L, Figure S12 ). Since Cd73-LNPs were found to achieve 100% complete remission in the ID8 model, we investigated the proportion of memory T cells present in the blood of ID8 model mice. The results showed that Cd73-LNPs had significantly increased the proportion of Cd44/Cd62l-positive Cd8 + T cells ( Figure S13 ). These results indicate that Cd73-LNPs enhance Cd8 + T cell infiltration into the tumor microenvironment, effectively suppress tumor growth, and demonstrate promising antitumor efficacy across multiple tumor models. Cd73-LNPs activated Cxcl/Cxcr signaling to promote Cd8 + T cell infiltration Activation and recruitment of CD8 + T cells to the tumor microenvironment are essential for effective anti-tumor responses. In our study utilizing the RM1-Psma model, we demonstrated that Cd73-LNPs enhanced immune cell infiltration into the tumor microenvironment and stimulated the secretion of effector molecules by Cd8 + T cells. Nonetheless, the precise mechanism by which Cd73-LNPs facilitated the recruitment of Cd8 + T cells to the tumor microenvironment remained to be elucidated. To gain a deeper understanding of the impact of Cd73-LNPs on T cells within the tumor immune microenvironment, we conducted single-cell RNA sequencing (scRNA-seq) on Cd45 + immune cells isolated from RM1-Psma xenografts harvested from C57BL/6J mice treated with either PBS or CD73-LNPs. Following quality control and filtering, we successfully captured a total of 12,327 cells, obtained their single-cell transcriptomes, and identified 21 transcriptionally homogeneous clusters using the t-distributed stochastic neighbor embedding (t-SNE) method (Figure S14A) . These clusters were annotated into 11 distinct cell types based on their transcriptional profiles and the SingleR package[15], including Cd3 + T cells, Cd4 + T cells, Cd8 + T cells, natural killer cells (NK), macrophages, monocytes, B cells, plasmacytoid dendritic cells (pDC), conventional dendritic cell 1 (cDC1), cDC2 and neutrophils. Notably, the proportion of Cd8 + T cells exhibited the most significant increase. (Fig. 5 A-B). In our study, we employed t-SNE analysis to categorize Cd8 + T cells into distinct subsets, including Cd8 + effector T cells, exhausted Cd8 + T cells, naïve Cd8 + T cells, early activated T cells, and tissue-resident memory Cd8 + T cells (Figure S14B-C) . Compared to mice treated with PBS, those treated with Cd73-LNPs exhibited an increased population of effector Cd8 + T cells and a decreased population of exhausted Cd8 + T cells (Figure S14A) . Additionally, volcano plots illustrating differentially expressed genes in Cd8 + T cells between the two groups corroborated the upregulation of genes associated with Cd8 + effector T cells, such as Gzma, Gzmb, and Gzmk (Figure S14D) . An analysis of intercellular communication networks revealed that Cxcl-related signaling was significantly upregulated in mice treated with Cd73-LNPs ( Fig. 5 C, S14E ) . The kinetics of chemokine expression within the tumor microenvironment (TME), along with alterations in chemokine receptor expression on immune cells, are likely determinants of the immune cell composition within the TME [24]. Subsequently, we conducted KEGG and GO enrichment analyses on the upregulated genes in intratumoral Cd8 + T cells. The results indicated enrichment in pathways related to lymphocyte activation and migration, chemokine signaling, and cytokine-cytokine receptor interactions ( Fig. 5 D-E ) . Chemokines facilitate directed cell migration by binding to chemokine receptors (CXCR). Consequently, we employed flow cytometry to assess the expression of CXCR receptors, which facilitate the migration of Cd8 + T cells in the blood, draining lymph nodes (DLN), and spleen. The findings demonstrated that post-Cd73-LNPs treatment, Cxcr3 expression was upregulated on Cd8 + T cells from the blood, DLN, and spleen; Cx3cr1 expression was upregulated on Cd8 + T cells from the blood and spleen; and Cxcr6 and Ccr5 expressions were upregulated exclusively in blood Cd8 + T cells ( Fig. 5 F-I, Figure S15-16) . The findings presented herein substantiate that Cd73-LNPs augment the migratory capacity of Cd8 + T cells through the activation of the Cxcl/Cxcr signaling pathway. Furthermore, Cd73-LNPs have been demonstrated to decrease the infiltration of exhausted T cells within the tumor microenvironment, underscoring their considerable therapeutic potential. Cd73-LNPs activated BMDCs through Tlr3 signaling Toll-like receptors (TLRs) initiate both shared and distinct signaling pathways that modulate immune responses[25]. Notably, among all TLRs, only the endosomal TLR3 does not trigger systemic inflammation and facilitates the cross-priming of antigen-specific CD8 + T cells by dendritic cells[26, 27]. TLR3 activation leads to downstream signaling cascades involving NF-kB, MAPKs, and IRF3, which result in the expression of inflammatory mediators and cytokines ( Fig. 6 A ) . We collected BMDCs which treated with Cd73-LNPs for 24 hours, transcriptome analysis was then performed via RNA sequencing. Subsequent transcriptome analysis was conducted using RNA sequencing. Principal component analysis (PCA) demonstrated robust replication within clusters and revealed significant differences between the Cd73-LNPs treatment group and PBS control group ( Fig. 6 B ) . The volcano plot shows that the expression of the genes associated with BMDC activation is significantly increased, including Ms4a6b, Slfn4, Fcgr1 (Fig. 6 C). The Gene Set Enrichment Analysis (GSEA) revealed a significant enrichment of the Toll-like receptor signaling pathway, specifically the TLR3 and TLR4 signaling pathways ( Fig. 6 D ) . Quantitative PCR (qPCR) experiments further confirmed that among the Toll-like receptors in mice, Tlr3 exhibited the most pronounced upregulation in expression following treatment with Cd73-LNPs ( Fig. 6 E ) . To ascertain whether the activation of BMDCs by Cd73-LNPs was contingent upon the Tlr3 signaling pathway, BMDCs were pretreated with small molecule inhibitors targeting Tlr3. Subsequently, the expression of cytokine genes and costimulatory molecules in BMDCs treated with Cd73-LNPs was analyzed. The findings demonstrated that CU CPT4a, a Tlr3-specific inhibitor, effectively inhibited the Cd73-LNPs-induced production of cytokines (Il-1β, Il-6, Ifn-α4, and Ifn-β1) and costimulatory molecules (Cd86, H-2m) in BMDCs ( Fig. 6 F-H, Figure S17). Subsequently, we collected BMDCs from Tlr3 −/− mice and observed that treatment with Cd73-LNPs significantly increased the phosphorylation levels of protein kinases, including NF-κB, mitogen-activated protein kinases (MAPKs), and Irf3, in wild-type (WT) BMDCs. However, this phosphorylation was substantially diminished in Tlr3 −/− BMDCs ( Fig. 6 I-J ) . Additionally, we assessed the therapeutic efficacy of Cd73-LNPs in subcutaneous tumor models of TC-1 and RM1-Psma using Tlr3 −/− mice. The tumor inhibitory effect of Cd73-LNPs was nearly completely nullified in Tlr3 −/− mice ( Fig. 6 K-N, Figure S18) . Furthermore, we conducted a comparative analysis of immune cell populations and proportions in the blood of wild-type and Tlr3 knockout mice using flow cytometry, which revealed no significant differences between the two groups (Figure S19) . These findings underscore the critical role of Tlr3 in mediating the antitumor effects induced by Cd73-LNPs. Collectively, the data strongly indicate that the antitumor efficacy of Cd73-LNPs necessitates both effective antigen expression and the activation of Tlr3 signaling pathways. Discussion In this study, the potential of Cd73 as a target for mRNA vaccines was investigated, with findings indicating that Cd73 mRNA vaccines effectively inhibit tumor growth in murine models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Mechanistically, the function of Cd73-LNPs was shown to depend on Tlr3 signaling in dendritic cells, leading to the upregulation of chemokine receptor expression on Cd8 + T cells. Furthermore, a comparative analysis was performed to evaluate the therapeutic efficacy of Cd73-LNPs in combination with a Cd73 monoclonal antibody (anti-Cd73) and the Cd73 inhibitor APCP in a prostate cancer model. The results revealed that APCP exhibited the greatest ability to inhibit adenosine metabolism in both serum and the tumor microenvironment. However, Cd73-LNPs were found to exert the most potent tumor-suppressive effect (Figure S20A-G ). These findings underscore the feasibility of utilizing Cd73 mRNA vaccines for the treatment of the aforementioned cancers, highlighting a promising strategy for cancer immunotherapy. The efficacy of mRNA vaccine approaches demonstrates notable variability, as most exhibit limitations related to the immunogenicity of the antigenic targets, the lack of systemic immune responses, and an inability to induce the rapid and adaptable immune responses that are likely essential for the effective suppression of rapidly evolving cancers[2]. Tumor-associated antigens have been demonstrated to exhibit innate immune tolerance, a factor which contributes to their insufficient immunogenicity[28, 29]. However, Cd73, as a tumor-associated antigen, has been shown to elicit potent anti-tumor immune responses. In the TC-1 model, the efficacy of the Cd73-LNPs was comparable to that of the TC-1-specific target mRNA vaccine E7-LNPs, with a CR of 60% (3/5). In the RM1-Psma model, Cd73-LNPs demonstrated significantly superior efficacy to Psma-LNPs, despite PSMA being considered a prostate cancer-specific target. The results of scRNA-seq indicated a significant increase in the proportion of effector T cells within the tumor microenvironment, coupled with a notable reduction in the proportion of exhausted T cells. The therapeutic efficacy of Cd73-LNPs is likely due to its dual mechanisms of activating anti-tumor immunity and mitigating the immunosuppressive state within the tumor microenvironment. Following administration of Cd73-LNPs, a notable enhancement in chemokine signaling and the infiltration of effector T cells within the tumor microenvironment was observed. Enrichment analysis further identified a significant enrichment of pathways associated with the activation and migration of Cd8 + T cells. Chemokines have been documented to facilitate the recruitment of circulating CD8 + T cells into the tumor microenvironment through interaction with their specific receptors[30]. The following chemokine receptors and their corresponding ligands are recognized as key mediators in the migration and activation of effector T cells: CXCR3 (CXCL9, CXCL10), CXCR1 (CXCL1), CXCR6 (CXCL16), and CCR5 (CCL5)[31]. The results of this study demonstrated an upregulation of all aforementioned receptors on the surface of Cd8 + T cells in the bloodstream, with a concurrent upregulation of Cxcr3 expression on Cd8 + T cells from the spleen and draining lymph nodes (DLN). Although the CXCR3/CXCL9-CXCL10 axis is also involved in the migration of regulatory T cells (Tregs)[32], in this study, it predominantly facilitated the migration of effector T cells. Moreover, this study demonstrated that Cd73-LNPs activate TLR3 signaling in dendritic cells, thereby enhancing antitumor immunity. Unlike most vaccines, which require adjuvants to boost immunogenicity, mRNA inherently possesses immunogenic properties and acts as a pathogen-associated molecular pattern. It exhibits affinity for various TLRs, such as TLR3, TLR7, and TLR8, as well as intracellular sensors[3, 33]. These characteristics facilitate the swift initiation of innate immune responses, in conjunction with the activation of adaptive immunity, as the mRNA payload is translated into full-length proteins and subsequently processed as antigens[3]. Conversely, lipid nanoparticles delivery systems have the potential to activate innate immune-related signaling pathways. For instance, the cationic lipids developed by Xia et al. have been demonstrated to activate Toll-like receptor 4. (TLR4) signaling in dendritic cells, thereby enhancing anti-tumor immunity[34]. Similarly, the lipids synthesized by Anderson et al. have been reported to activate the stimulator of interferon genes (STING) signaling pathway, thus augmenting immune responses in vivo[35]. The mRNA delivery system utilized in this study consisted of LNPs previously engineered to induce cytokine secretion in bone marrow-derived dendritic cells (BMDCs), although the precise mechanism remains to be elucidated. In this investigation, Luc-LNPs were observed to induce a moderate upregulation of Tlr3 expression in BMDCs, whereas Cd73-LNPs demonstrated a more substantial upregulation of Tlr3. This suggests that the activation of Tlr3 by Cd73-LNPs likely represents a synergistic interaction between the delivery system and Cd73 mRNA. This study is limited by the lack of investigation into the specific mechanism underlying the ineffectiveness of Cd73-LNPs treatment following Tlr3 deficiency. Furthermore, Cd8 + T cell chemokine receptor expression was only validated in the RM1 model following Cd73-LNPs treatment. Moreover, the therapeutic efficacy of Cd73-LNPs was not explored further in humanized mice or other models. In conclusion, we have demonstrated that the Cd73 protein is highly immunogenic, the Cd73 mRNA vaccine has been shown to safely and effectively induce antitumor immunity, suggesting significant potential for clinical translation. Abbreviations LNPs: Lipid nanoparticles, NK: Natural killer, BMDCs: Bone marrow-derived dendritic cells, PDI: Polydispersity index, ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, BUN: Blood urea nitrogen, CREAT: creatinine. Gra B: granzyme B, TLR: toll-like receptor. Declarations Ethics approval and consent to participate: The Institutional Animal Care and Use Committee (IACUC) at Sun Yat-Sen University has formally approved all the animal procedures (SYSU-IACUC-2025-002827). Consent for publication: All authors have read and approved the manuscript. Availability of data and material: All data are included in the paper or the supplemental information. Additional data are available from the corresponding authors on reasonable request. Competing interests: The authors declare no competing interests. Funding: This work was supported by Shenzhen Fundamental Research Program (Grant No. JCYJ20240813150249044, JCYJ20250604143756074), Guangdong Basic and Applied Basic Research Foundation (Grant No. 2023A1515030058), the National Natural Science Foundation of China (Grant No. 82272689). Authors’ contributions: Yuandong Xu, Fei Cao, Ze-Xiu Xiao, Gao-feng Zha, and Jun Pang. developed this concept and designed this study. Yuandong Xu, Fei Cao, Haowei Qiu, Yiting Zhang, Zhen Xu, Yunru He performed the experiments and acquired the data. Yuandong Xu and Jun Pang acquired the funding. Yuandong Xu, Fei Cao, Haowei Qiu, and Yi Zhang performed data analysis. Yuandong Xu and Yi Zhang performed single-cell transcriptome sequencing and RNA sequencing analysis. Yuandong Xu, Ze Xiu Xiao, Gao-feng Zha, and Jun Pang edited and revised the manuscript. Acknowledgements: It is imperative to express our profound gratitude to the dedicated staff at Shenzhen MagicRNA Biotech for their invaluable support during the implementation of this study. References Lorentzen CL, Haanen JB, Met Ö, Svane IM. Clinical advances and ongoing trials on mRNA vaccines for cancer treatment. Lancet Oncol. 2022; 23: e450-e8. Yaremenko AV, Khan MM, Zhen X, Tang Y, Tao W. Clinical advances of mRNA vaccines for cancer immunotherapy. Med. 2025; 6: 100562. Li H, Min L, Du H, Wei X, Tong A. Cancer mRNA vaccines: clinical application progress and challenges. Cancer Lett. 2025; 625: 217752. Floudas CS, Sarkizova S, Ceccarelli M, Zheng W. Leveraging mRNA technology for antigen based immuno-oncology therapies. J Immunother Cancer. 2025; 13. Sayour EJ, Boczkowski D, Mitchell DA, Nair SK. Cancer mRNA vaccines: clinical advances and future opportunities. Nat Rev Clin Oncol. 2024; 21: 489-500. Cui M, Ma S, Huang Z, Zhang D, Sun X, You Y. Medicinal Chemistry Strategies for the Development of CD73 Inhibitors in Cancer Immunotherapy. Med Res Rev. 2025. Shen J, Liao B, Gong L, Li S, Zhao J, Yang H, et al. CD39 and CD73: biological functions, diseases and therapy. Mol Biomed. 2025; 6: 97. Zhang H, Yang L, Han M, Han Y, Jiang Z, Zheng Q, et al. Boost Infiltration and Activity of T Cells via Inhibiting Ecto-5'-nucleotidase (CD73) Immune Checkpoint to Enhance Glioblastoma Immunotherapy. ACS Nano. 2024; 18: 23001-13. Deng Y, Chen Q, Yang X, Sun Y, Zhang B, Wei W, et al. Tumor cell senescence-induced macrophage CD73 expression is a critical metabolic immune checkpoint in the aging tumor microenvironment. Theranostics. 2024; 14: 1224-40. Bodogai M, Park B, Braikia FZ, Naqing F, Kumaraswami K, Chen C, et al. A distinct population of CD8(+) T cells expressing CD39 and CD73 accumulates with age and supports cancer progression. Nat Aging. 2025; 5: 2055-69. Neo SY, Yang Y, Record J, Ma R, Chen X, Chen Z, et al. CD73 immune checkpoint defines regulatory NK cells within the tumor microenvironment. J Clin Invest. 2020; 130: 1185-98. Klysz DD, Fowler C, Malipatlolla M, Stuani L, Freitas KA, Chen Y, et al. Inosine induces stemness features in CAR-T cells and enhances potency. Cancer Cell. 2024; 42: 266-82.e8. Xu Y, Hu Y, Xia H, Zhang S, Lei H, Yan B, et al. Delivery of mRNA Vaccine with 1, 2-Diesters-Derived Lipids Elicits Fast Liver Clearance for Safe and Effective Cancer Immunotherapy. Adv Healthc Mater. 2024; 13: e2302691. Cao F, Xu Y, Guan Y, Zhang K, Qiu H, Xu Z, et al. Enhancing the potency of 5T4 mRNA vaccine by CD70 mRNA-LNPs through ADCC and T cell boosting in prostate cancer therapy. J Nanobiotechnology. 2025; 23: 523. Sun Y, Zhang Z, Li H, Bu X, Chen L, Wang X, et al. Destruction of VISTA by TRIM25 ablation in T cells potentiates cancer immunotherapy. Cell Res. 2025; 35: 1003-20. Huber F, Bassani-Sternberg M. Defects in antigen processing and presentation: mechanisms, immune evasion and implications for cancer vaccine development. Nat Rev Immunol. 2025. Lee CYC, Kennedy BC, Richoz N, Dean I, Tuong ZK, Gaspal F, et al. Tumour-retained activated CCR7(+) dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity. Nat Commun. 2024; 15: 682. Gosselin EA, Eppler HB, Bromberg JS, Jewell CM. Designing natural and synthetic immune tissues. Nat Mater. 2018; 17: 484-98. He X, Wang J, Tang Y, Chiang ST, Han T, Chen Q, et al. Recent Advances of Emerging Spleen-Targeting Nanovaccines for Immunotherapy. Adv Healthc Mater. 2023; 12: e2300351. Xu Z, Ma W, Wang J, Chen H, Li H, Yin Z, et al. Nuclear HMGB1 is critical for CD8 T cell IFN-γ production and anti-tumor immunity. Cell Rep. 2024; 43: 114591. van der Sluis TC, van Haften FJ, van Duikeren S, Pardieck IN, de Graaf JF, Vleeshouwers W, et al. Delayed vaccine-induced CD8(+) T cell expansion by topoisomerase I inhibition mediates enhanced CD70-dependent tumor eradication. J Immunother Cancer. 2023; 11. Bakht MK, Beltran H. Biological determinants of PSMA expression, regulation and heterogeneity in prostate cancer. Nat Rev Urol. 2025; 22: 26-45. Hage Chehade C, Ozay ZI, Ostrowski M, Mercinelli C, Gebrael G, Sayegh N, et al. T-cell Engagers in Prostate Cancer. Eur Urol. 2025; 87: 553-8. Mempel TR, Lill JK, Altenburger LM. How chemokines organize the tumour microenvironment. Nat Rev Cancer. 2024; 24: 28-50. Kawai T, Ikegawa M, Ori D, Akira S. Decoding Toll-like receptors: Recent insights and perspectives in innate immunity. Immunity. 2024; 57: 649-73. Koerner J, Horvath D, Herrmann VL, MacKerracher A, Gander B, Yagita H, et al. PLGA-particle vaccine carrying TLR3/RIG-I ligand Riboxxim synergizes with immune checkpoint blockade for effective anti-cancer immunotherapy. Nat Commun. 2021; 12: 2935. Matsumoto M, Takeda Y, Tatematsu M, Seya T. Toll-Like Receptor 3 Signal in Dendritic Cells Benefits Cancer Immunotherapy. Front Immunol. 2017; 8: 1897. Fan T, Zhang M, Yang J, Zhu Z, Cao W, Dong C. Therapeutic cancer vaccines: advancements, challenges, and prospects. Signal Transduct Target Ther. 2023; 8: 450. Leko V, Rosenberg SA. Identifying and Targeting Human Tumor Antigens for T Cell-Based Immunotherapy of Solid Tumors. Cancer Cell. 2020; 38: 454-72. Yi M, Li T, Niu M, Zhang H, Wu Y, Wu K, et al. Targeting cytokine and chemokine signaling pathways for cancer therapy. Signal Transduct Target Ther. 2024; 9: 176. Ozga AJ, Chow MT, Luster AD. Chemokines and the immune response to cancer. Immunity. 2021; 54: 859-74. Moreno Ayala MA, Campbell TF, Zhang C, Dahan N, Bockman A, Prakash V, et al. CXCR3 expression in regulatory T cells drives interactions with type I dendritic cells in tumors to restrict CD8(+) T cell antitumor immunity. Immunity. 2023; 56: 1613-30.e5. Cao LL, Kagan JC. Targeting innate immune pathways for cancer immunotherapy. Immunity. 2023; 56: 2206-17. Zhang H, You X, Wang X, Cui L, Wang Z, Xu F, et al. Delivery of mRNA vaccine with a lipid-like material potentiates antitumor efficacy through Toll-like receptor 4 signaling. Proc Natl Acad Sci U S A. 2021; 118. Miao L, Li L, Huang Y, Delcassian D, Chahal J, Han J, et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat Biotechnol. 2019; 37: 1174-85. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8715323","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":582580774,"identity":"8d6c5934-8c19-419f-a79e-6a3bb39fb026","order_by":0,"name":"Yuandong Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYJACiQ8VNgwMzMwNMAEDglokZ5xJA2phJEGLNG/bYSBFrBaDGzmGN3jOnI/mb2dsYPzZVpfYwN68TYKh5g4eR+UYW0hU3M6dcZixgRloXWIDz7EyCYZjz3Bq4ZfIMZMwOHM7twGkhbHtQGIDSISx4TBOLWwgBYlt53LnH4Y5TP4Nfi1gWw62HcjdANTCwNvGDLSFB78WyZ5nxZYNZ5JzNwK1HOY5d9i4jSet2CLhGG4tBseTN97+U2GXO+/84YMPf5TVyfazH95440MNbi0MDByIWDjAyAb0HYiVgEcDAwP7AyTOH7xKR8EoGAWjYIQCAP0aWGypNUL9AAAAAElFTkSuQmCC","orcid":"","institution":"The Seventh Affiliated Hospital Sun Yat-sen University","correspondingAuthor":true,"prefix":"","firstName":"Yuandong","middleName":"","lastName":"Xu","suffix":""},{"id":582580775,"identity":"00bd19ba-1a74-4b23-b020-c70643619d5a","order_by":1,"name":"Fei Cao","email":"","orcid":"","institution":"The Seventh Affiliated Hospital Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Cao","suffix":""},{"id":582580776,"identity":"aeeecd36-ac7c-4885-be0b-17ff13fc65ce","order_by":2,"name":"Haowei Qiu","email":"","orcid":"","institution":"The Seventh Affiliated Hospital Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Haowei","middleName":"","lastName":"Qiu","suffix":""},{"id":582580777,"identity":"1371aac6-6d90-418b-85de-33f7da9a5ae2","order_by":3,"name":"Yi Zhang","email":"","orcid":"","institution":"The Seventh Affiliated Hospital Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":582580778,"identity":"1f4deb75-171d-459e-9367-6dbe93653507","order_by":4,"name":"Yiting Wang","email":"","orcid":"","institution":"The Seventh Affiliated Hospital Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Yiting","middleName":"","lastName":"Wang","suffix":""},{"id":582580779,"identity":"81a4b7c1-1486-4ac6-88df-2321edd23994","order_by":5,"name":"Zhen Xu","email":"","orcid":"","institution":"The Seventh Affiliated Hospital Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Xu","suffix":""},{"id":582580780,"identity":"3ce4ac2f-d5e6-4bf8-9ab0-c17da458288f","order_by":6,"name":"Yunru He","email":"","orcid":"","institution":"The Seventh Affiliated Hospital Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Yunru","middleName":"","lastName":"He","suffix":""},{"id":582580781,"identity":"219d3876-eb14-4f0c-afdb-35f766746af6","order_by":7,"name":"Ze-Xiu Xiao","email":"","orcid":"","institution":"Shenzhen MagicRNA Biotech","correspondingAuthor":false,"prefix":"","firstName":"Ze-Xiu","middleName":"","lastName":"Xiao","suffix":""},{"id":582580782,"identity":"21a5b0fe-a1f5-4efc-ab0f-be2269dbaa6c","order_by":8,"name":"Gao-Feng Zha","email":"","orcid":"","institution":"Shenzhen MagicRNA Biotech","correspondingAuthor":false,"prefix":"","firstName":"Gao-Feng","middleName":"","lastName":"Zha","suffix":""},{"id":582580783,"identity":"a4b13276-0e4b-4106-af77-7abf5618047c","order_by":9,"name":"Jun Pang","email":"","orcid":"https://orcid.org/0000-0003-0024-9415","institution":"The Seventh Affiliated Hospital Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Pang","suffix":""}],"badges":[],"createdAt":"2026-01-28 02:10:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8715323/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8715323/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101649485,"identity":"d47e586f-94d6-494e-973c-256e76f520e5","added_by":"auto","created_at":"2026-02-02 09:10:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":486545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSafety profile and in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e immunogenicity of Cd73 protein induced by Cd73-LNPs.\u003c/strong\u003e (A) Schematic diagram of Cd73-LNPs. (B) Capillary electrophoresis analysis Gel image of Cd73 mRNA solution (1 µg µL\u003csup\u003e−1\u003c/sup\u003e). (C) Average size and representative cryogenic electron microscopy images of Cd73-LNPs. (D-F) Western blot and Flow cytometry analysis (mean±SD, n=3) of Cd73 protein expression induced by Cd73-LNPs transfection in BMDCs. β-actin serves as a loading control. (G) CCK-8 assay was employed to evaluate the effects of different concentrations of Cd73-LNPs on BMDCs viability (mean±SD, n=4). (H) ALT, AST, CREA, and CK blood testing results after Cd73-LNPs immunization (mean±SD, n=5). (I) IFN-γ spot-forming cells (J) and statistical data (K) from restimulated splenocytes determined by the ELISPOT assay on day 10 after Cd73-LNPs immunization. Data indicate mean ± SD of biological replicates (n=5 per group). Error bars represent 95% CI. ****p \u0026lt; 0.0001; ***p \u0026lt; 0.001; **p \u0026lt; 0.01; *p \u0026lt; 0.05; ns., not significant. ALT: Alanine Aminotransferase, AST: Aspartate Aminotransferase, CREA: Creatinine, CK: Creatine Kinase.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/da6fadef6c336981a2843efe.png"},{"id":101649470,"identity":"5e85cc43-525e-4ffc-b2d3-4f23d7d562e9","added_by":"auto","created_at":"2026-02-02 09:10:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":447461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCd73-LNPs induced the maturation and activation of BMDCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic Diagram of dendritic cell-mediated T cell activation. (B) The mRNA levels of cytokines including I\u003cem\u003el-1β\u003c/em\u003e, I\u003cem\u003el-6\u003c/em\u003e, I\u003cem\u003el-12α\u003c/em\u003e, I\u003cem\u003el-12p40\u003c/em\u003e, I\u003cem\u003efn-α1\u003c/em\u003e, I\u003cem\u003efn-α4\u003c/em\u003e and I\u003cem\u003efn-β\u003c/em\u003e in BMDCs induced by Cd73-LNPs (mean±SD, n=3), mRNA: 1ug/uL, Control: PBS buffer. treatment time: 24 h.(C) Protein levels of Il-1β, Il-6, Il-12p70 and Ifn-β in culture media of BMDCs by treatments as in B, measured by ELISA (mean±SD, n=3). Treatment time: 24 h.(D-I) Flow cytometry analysis and corresponding statistical graphs depicting the proportions of Cd80\u003csup\u003e+\u003c/sup\u003e(D), Cd86\u003csup\u003e+\u003c/sup\u003e(E), Ox40l\u003csup\u003e+\u003c/sup\u003e(F), Cd103\u003csup\u003e+\u003c/sup\u003e(G), 4-1bbl\u003csup\u003e+\u003c/sup\u003e(H), Ccr7\u003csup\u003e+\u003c/sup\u003e (I)BMDCs (mean±SD, n=3). Error bars represent 95% CI. ****p \u0026lt; 0.0001; ***p \u0026lt; 0.001; **p \u0026lt; 0.01; *p \u0026lt; 0.05; ns., not significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/d8ab08c6db0e1966a1b9d0a6.png"},{"id":101649340,"identity":"d0e48a3c-18ca-45b3-8f38-636a5b511c3c","added_by":"auto","created_at":"2026-02-02 09:09:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":464102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCd73-LNPs activated potent humoral and cellular immunity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) The timeline and experimental groups for Cd73-LNPs administration in tumor-free mice. (B) Relative levels of adenosine (left), Cd73 protein (middle), and anti-Cd73 IgG (right) in serum, measured by ELISA (mean±SD, n=5). (C-D) Flow cytometry analysis of the proportion of different immune cells in the spleen, including Cd3\u003csup\u003e+\u003c/sup\u003e T cell, B cell, NK cell, DC, macrophage, Cd4\u003csup\u003e+\u003c/sup\u003e T cell, Cd8\u003csup\u003e+\u003c/sup\u003e T cell (mean±SD, n=5). (E) Flow cytometry analysis and corresponding statistical graphs depicting the proportions of B220\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;/CD138\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;B cells in spleen (mean±SD, n=5). (F-I) Flow cytometry analysis and statistical graphs showing the proportions of Ifn-γ \u003csup\u003e+\u003c/sup\u003e(F), Gra B\u003csup\u003e+\u003c/sup\u003e(G), Tnf-α\u003csup\u003e+\u003c/sup\u003e (H), Eomes\u003csup\u003e+\u003c/sup\u003e (I) in Cd8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells in spleen (mean±SD, n=5). Error bars represent 95% CI. ****p \u0026lt; 0.0001; ***p \u0026lt; 0.001; **p \u0026lt; 0.01; *p \u0026lt; 0.05; ns., not significant. DC: Dendritic cells, Gra B: Granomycin B.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/3dfb818553a4886e3dc64880.png"},{"id":101650084,"identity":"31293ab9-6ffa-4107-aa7d-67fd2f42c142","added_by":"auto","created_at":"2026-02-02 09:12:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":433292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCd73-LNPs improved immune cell infiltration in the tumor microenvironment and enhanced anti-tumor efficacy.\u003c/strong\u003e (A)\u0026nbsp;Schematic of the experiment protocol. (B-I) The tumor size (n=5) and overall survival (n=5) in B16-OVA model, 5×10\u003csup\u003e5\u003c/sup\u003e/ cells/mouse (B-C), TC-1 model, 5×10\u003csup\u003e5\u003c/sup\u003e/ cells/mouse (D-E), ID8 model, 1×10\u003csup\u003e6\u003c/sup\u003e/ cells/mouse (F-G), RM1-Psma model, 2.5×10\u003csup\u003e5\u003c/sup\u003e/ cells/mouse (H-I). (J) Flow cytometry analysis of the proportion of different immune cells in the tumor microenvironment (mean±SD, n=5), including Cd45\u003csup\u003e+\u003c/sup\u003e cell, Cd8\u003csup\u003e+\u003c/sup\u003e T cell, Cd4\u003csup\u003e+\u003c/sup\u003e T cell, NK cell. (K-L) Flow cytometry analysis and statistical graphs showing the proportions of Ifn-γ \u003csup\u003e+\u003c/sup\u003e, Gra B\u003csup\u003e+\u003c/sup\u003e in Cd8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells in tumor microenvironment (mean±SD, n=5). Error bars represent 95% CI. ****p \u0026lt; 0.0001; ***p \u0026lt; 0.001; **p \u0026lt; 0.01; *p \u0026lt; 0.05; ns., not significant. CR: Complete remission rate.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/97341c542a120cb01b83dd88.png"},{"id":101650157,"identity":"1a4ab5f3-2ddb-4298-9148-a49754c65817","added_by":"auto","created_at":"2026-02-02 09:12:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":647647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCd73-LNPs activated Cxcl/Cxcr signaling to promote Cd8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell infiltration. \u003c/strong\u003e(A) The t-distributed stochastic neighbour embedding (t-SNE) plot of the 11 identified main cell types in subcutaneous RM1-Psma tumors (n=3). (B) Dot plots showing expression patterns of conventional marker genes in different immune cell types. (C) Analysis of intercellular communication networks between PBS groups and Cd73-LNPs groups. (D-E) GO enrichment analysis and KEGG pathway enrichment analysis of Cd8\u003csup\u003e+\u003c/sup\u003e T cell. (F-I) Flow cytometry analysis and statistical graphs showing the proportions of Cxcr3\u003csup\u003e+\u003c/sup\u003e(H), Cx3cr1\u003csup\u003e+\u003c/sup\u003e(I), Cxcr\u003csup\u003e+\u003c/sup\u003e(J) and Ccr5\u003csup\u003e+\u003c/sup\u003e(I) cells in Cd8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells from blood (mean±SD, n=5). Error bars represent 95% CI. ****p \u0026lt; 0.0001; ***p \u0026lt; 0.001; **p \u0026lt; 0.01; *p \u0026lt; 0.05; ns., not significant.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/c21cd95b2d3d8c62cdbceadf.png"},{"id":101649966,"identity":"9304ebbc-1d9b-45a1-bc10-c411bb33a339","added_by":"auto","created_at":"2026-02-02 09:11:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":545304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCd73-LNPs activated BMDCs through Tlr3 signaling \u003c/strong\u003e(A) Schematic diagram of the Tlr3 signaling pathway. (B) Principal component analysis (PCA) revealed that Cd73-LNPs treated BMDCs formed separate clusters from the control mice group (n=3). (C) Volcano plot showing differential gene expression in BMDCs (n=3). (D) Gene set enrichment analysis of BMDCs in Cd73-LNPs vs. PBS groups. (E) The mRNA expression of different toll like receptor in BMDCs from Cd73-LNPs, Luc-LNPs or PBS groups (n=3). (F) The mRNA levels of cytokines including I\u003cem\u003el-1β\u003c/em\u003e, I\u003cem\u003el-6\u003c/em\u003e, I\u003cem\u003el-12α\u003c/em\u003e, I\u003cem\u003el-12p40\u003c/em\u003e, I\u003cem\u003efn-α1\u003c/em\u003e, I\u003cem\u003efn-α4\u003c/em\u003e and I\u003cem\u003efn-β\u003c/em\u003e1 in BMDCs from Cd73-LNPs, Cd73-LNPs + Tlr3 inhibitor (pre-treated 1h) or PBS (mean±SD, n=3), mRNA: 1ug/uL, treatment time: 24 h. (G) Flow cytometry analysis and corresponding statistical graphs depicting the proportions of Cd80\u003csup\u003e+\u003c/sup\u003e, H-2m\u003csup\u003e+\u003c/sup\u003e BMDCs (mean±SD, n=3). \u003cstrong\u003e(I-J) \u003c/strong\u003e(K-N) Antitumor efficacy of the Cd73-LNPs on WT and\u0026nbsp;\u003cem\u003eTlr3\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e mice (TC-1 and RM1-Psma), n=3. Error bars represent 95% CI. ****p \u0026lt; 0.0001; ***p \u0026lt; 0.001; **p \u0026lt; 0.01; *p \u0026lt; 0.05; ns., not significant.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/8482b3e34d6ab8410656cfa8.png"},{"id":102298652,"identity":"f72ba0d2-b382-42b1-a1a9-be0f19daedca","added_by":"auto","created_at":"2026-02-10 10:56:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4237623,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/bf0cdb5a-3780-408f-9db3-8924595ba63c.pdf"},{"id":101649586,"identity":"c9dfe398-7785-4f01-aed1-64a2920c04eb","added_by":"auto","created_at":"2026-02-02 09:10:55","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14297712,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/a3302e0b81bd28e972163f5a.docx"},{"id":101650117,"identity":"96e50956-2146-4fca-8eb4-01443c65e8c1","added_by":"auto","created_at":"2026-02-02 09:12:19","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":371072,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8715323/v1/667a8f54f644148dd377bfd1.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eCd73-LNPs promotes antitumor T-cell immunity via amplifying Tlr3-mediated immunostimulatory dendritic cell activation\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003emRNA vaccines have emerged as a promising platform for cancer immunotherapy, characterized by high potency, safe administration, rapid development potential, and cost-effective manufacturing[1]. These vaccines function by encoding tumor-specific antigens or costimulatory molecules, which are subsequently translated into proteins by the patient's cells, particularly antigen-presenting cells, thereby eliciting a robust anti-tumor immune response [2\u0026ndash;4]. Despite significant advancements in the field of cancer mRNA vaccines, challenges persist, particularly concerning the poor immunogenicity of antigenic targets and the inability to induce rapid yet adaptable immune responses, which are likely necessary to suppress rapidly evolving cancers[5]. Identifying immunogenic antigenic targets holds substantial potential for accelerating the clinical implementation of mRNA vaccines.\u003c/p\u003e \u003cp\u003eCD73, a membrane-bound ecto-5'-nucleotidase, facilitates the extracellular conversion of adenosine monophosphate into the immunosuppressive molecule adenosine[6]. Its upregulation is frequently observed across various tumor types, including melanoma, ovarian cancer, and prostate cancer, thereby contributing to the accumulation of adenosine within the tumor microenvironment and promoting immune evasion[7]. In addition to its enzymatic activity, CD73 plays a critical role in cell adhesion and migration in cancers[8]. Moreover, CD73 expression has been detected on a range of immune cells, such as T cells, macrophages, and natural killer cells, and its involvement in the immunosuppressive functions of these cells has been elucidated[9\u0026ndash;11]. Given the evidence of CD73's immunosuppressive and pro-tumor effects, the targeted inhibition of the CD73 pathway has emerged as a highly promising therapeutic strategy. Significant efforts have been devoted to the development of CD73-targeting therapeutics, such as small-molecule inhibitors, monoclonal antibodies, and bispecific antibodies[6, 12]. Nonetheless, no such drug has yet advanced to the clinical stage.\u003c/p\u003e \u003cp\u003eOur previous research developed lipid nanoparticle (LNP) delivery systems specifically targeting the spleen and draining lymph nodes [13]. In the current study, building upon our prior work with LNPs, we examined the therapeutic efficacy and underlying mechanisms of Cd73 mRNA vaccines (Cd73-LNPs) in a mouse model. The findings demonstrated that Cd73-LNPs elicit robust antitumor immune responses in vivo through antigen presentation pathways, facilitating the secretion of effector molecules such as Ifn-γ and granzyme B (Gra B) by Cd8\u003csup\u003e+\u003c/sup\u003e T cells, and effectively inhibiting tumor growth in models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Mechanistically, Cd73-LNPs were shown to upregulate chemokine receptor expression on Cd8\u003csup\u003e+\u003c/sup\u003e T cell surfaces, thereby enhancing Cd8\u003csup\u003e+\u003c/sup\u003e T cell infiltration into the tumor microenvironment. Additionally, our research indicated that Cd73-LNPs activate dendritic cells by stimulating the Toll-like receptor 3 (Tlr3) signaling pathway, thereby promoting dendritic cell activation. This study underscores the strong immunogenicity of the Cd73 protein and highlights the significant potential of Cd73 mRNA vaccines for clinical translation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCulture of cell lines\u003c/h2\u003e \u003cp\u003eThe B16-OVA, TC-1, ID8, and RM-1 cells were cultivated in RPMI-1640 medium (Gibco, USA) with the addition of 10% foetal calf serum (Gibco, USA) and 1% Penicillin-Streptomycin Solution (Pricella, USA). All cell lines were obtained from the American Type Culture Collection (ATCC) website. A regular programme of checks was implemented for the purpose of verifying the absence of mycoplasma contamination in all cell lines. The methodology employed for this purpose was the MycAwayTM Plus-Color One-Step Mycoplasma Detection Kit (YEASEN, China).\u003c/p\u003e \u003cp\u003eThe isolation of bone marrow-derived dendritic cells (BMDCs) was achieved by harvesting bone marrow from the femurs and tibias of C57BL/6J mice. Following red blood cell lysis using ACK lysis buffer, the remaining bone marrow cells were cultured in RPMI 1640 medium, which was supplemented with 10% foetal bovine serum (FBS), 1% penicillin\u0026ndash;streptomycin, 20 ng/mL recombinant GM-CSF (rm-GM-CSF, Peprotech, USA) and 20 ng/mL recombinant IL-4 (rm-IL-4, Peprotech, USA). This was done to promote dendritic cell differentiation. The medium was replaced at two-day intervals, with half of it being replaced in order to maintain optimal growth conditions. On the sixth day, the immature BMDCs were harvested from the culture dish and utilised in subsequent experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003emRNA synthesis\u003c/h3\u003e\n\u003cp\u003eThe transcription of Cd73 mRNA, E7 mRNA, Psma mRNA and Luciferase mRNA was conducted in vitro, utilising T7 RNA polymerase (Megascript, Ambion, USA) and linearized plasmid templates. In the transcription reaction, methylated pseudouridine (m1Ψ)-5\u0026prime;-triphosphate (TriLink) was utilised as a substitute for the conventional uridine (UTP), thereby introducing modifications to the mRNA nucleotides. Subsequently, the synthesised mRNA was 5\u0026prime;-capped using 2\u0026prime;-O-methyltransferase (ScriptCap; CellScript, USA) and an m7G capping kit to enhance its stability and translation efficiency. The synthesis of mRNA was followed by a purification process, which involved the use of a cellulose column (Sigma-Aldrich, USA), with the aim of ensuring the removal of any impurities. The synthesis of mRNA was subjected to rigorous quality control measures. This was achieved by means of capillary electrophoresis, utilising the Agilent Technologies 5200 Fragment Analyzer (Agilent Technologies, USA). The integrity of the mRNA was assessed using the DNF-472-1000 kit and gel retardation method. The synthetic mRNA samples were stored at -80\u0026deg;C until further use.\u003c/p\u003e\n\u003ch3\u003emRNA-LNPs Preparation and characterization\u003c/h3\u003e\n\u003cp\u003eThe ionizable amino lipid (E12A1A3) synthesis and complete methodology for mRNA-LNPs synthesis have been thoroughly described in our previous study[13]. The organic phase dissolved in ethanol (the ethanol phase) at a molar ratio of E12A1A3/DSPC (Avanti. China)/cholesterol (Avanti. China)/DMG-PEG 2000 (Avanti. China) of 50:10:38.5:1.5. Meanwhile, the mRNA was dissolved in sodium citrate solution (the aqueous phase). The ethanol and aqueous phases were combined at a volume ratio of 1:3, with a lipid-to-mRNA N/P ratio of 5:1. The subsequent step involved the amalgamation of lipids and mRNA, which was achieved by means of a syringe pump within a microfluidic chip device (INanoE, MicroNano, China). The final product was then subjected to a centrifugal process at 1200 \u0026times; g for 10 minutes, utilising a 100 kDA ultrafiltration membrane. This was followed by a thorough washing procedure and resuspension in Phosphate Buffered Saline (PBS). The preparations were stored at a temperature of 4\u0026deg;C. The particle size distribution of the mRNA-LNPs was analysed using dynamic light scattering (DLS) on a Zetasizer Nano (Malvern Instruments, Malvern, UK). The Quant-iT RiboGreen RNA Assay Kit (Invitrogen, USA) was utilised to evaluate concentration and encapsulation efficiency.\u003c/p\u003e \u003cp\u003e \u003cb\u003emRNA-LNPs cytotoxicity test in\u003c/b\u003e \u003cb\u003evitro\u003c/b\u003e \u003cb\u003eand in\u003c/b\u003e \u003cb\u003evivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe BMDCs were then exposed to different concentrations of mRNA-LNPs or PBS for 24 hours. After this, the BMDCs were collected and the number of living cells was measured using a kit called the Cell Counting Kit-8 (CCK8) (Beyotime, China). In short, the cells were put in a 96-well plate at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u0026ndash;1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well and left to settle for overnight. After 24 hours, the cells were treated with different concentrations of mRNA-LNPs or PBS as a control. After treatment, 10 \u0026micro;L of CCK8 solution was added to each well and the plate was left for two more hours at 37\u0026deg;C. The amount of light absorbed at 450 nm was measured using a special machine, and the number of living cells was calculated as a percentage of the control group. We used this formula to work out how many cells were alive: Relative viability (%) = [(OD\u003csub\u003e_treated\u003c/sub\u003e \u0026minus; OD\u003csub\u003e_blank)\u003c/sub\u003e / (OD\u003csub\u003e_control\u003c/sub\u003e \u0026minus; OD\u003csub\u003e_blank\u003c/sub\u003e)] \u0026times; 100.\u003c/p\u003e \u003cp\u003eSerum samples were collected from tumor-free mice following Cd73-LNPs administration for biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and Creatine Kinase (CK), undertake a comprehensive evaluation of hepatic and renal function.\u003c/p\u003e\n\u003ch3\u003eReal-Time PCR\u003c/h3\u003e\n\u003cp\u003eThe TRIzol Reagent (Invitrogen) was utilised for the isolation of total cellular RNA, in accordance with the manufacturer's instructions. The synthesis of cDNA was accomplished through the employment of the Hifair II 1st Strand cDNA Synthesis Kit (Yeasen). The quantification of target mRNA expression was performed using the Hieff UNICON Universal Blue qPCR SYBR Green Master Mix Kit (Yeasen). Subsequent analysis of the results was conducted within the Bio-Rad detection system. The relative target mRNA expression was calculated by using a 2ˆ-ΔΔCT method and normalized to the expression of \u0026#120573;-actin. The primer sequences utilised in the present study as showed in previous article and Table\u0026nbsp;2[13].\u003c/p\u003e\n\u003ch3\u003eEnzyme-Linked Immunosorbent Assays (ELISA)\u003c/h3\u003e\n\u003cp\u003eRelative ELISA kits were procured from eBioscience with the objective of detecting the cytokines (Il-1\u0026#120573;, Il-2, Il-6, and Il-12P70) released from BMDCs culture supernatant.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eELispot assay for Cd73-LNPs\u003c/h2\u003e \u003cp\u003eEnzyme-Linked Immunospot (ELISpot): IFN-\u0026#120574; production by splenocytes isolated from post-immunized mice was measured by ELISpot. The ELISpot was performed strictly as described[13]. Bone marrow-derived dendritic cells (BMDCs) were differentiated from C57BL/6J mice and treated with Cd73-LNPs for a period of 24 hours. T cells were isolated from the spleens of C57BL/6J mice and co-cultured with mRNA-treated BMDCs at a ratio of 1:10 for a further 24 to 48 hours. Following the incubation period, IFN-γ secretion by activated T cells was quantified using an IFN-γ ELISpot assay (Dakewe, China). The wells were pre-coated with an anti-IFN-γ capture antibody, and after incubation, spot formation was visualised using a biotinylated detection antibody and streptavidin-alkaline phosphatase conjugate, eflecting its immunogenic potential.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal experiment\u003c/h3\u003e\n\u003cp\u003e The Institutional Animal Care and Use Committee (IACUC) at Sun Yat-Sen University has formally approved all the animal procedures (SYSU-IACUC-2025-002827). All animal experiments were performed under pathogen-free conditions, in compliance with the Laboratory Animal Care and Use Guidelines set by the National Institute of Health. C57BL/6 mice, aged between 6 and 8 weeks, were procured from Gemmatech, Tlr3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice purchased from Cyagen. The subcutaneous tumor models employed in the present study included B16-OVA, TC-1, ID8, and RM-1-Psma, with tumor cell inoculation doses of 5x10\u003csup\u003e5\u003c/sup\u003e, 5x10\u003csup\u003e5\u003c/sup\u003e, 1x10\u003csup\u003e6\u003c/sup\u003e and 2.5x10\u003csup\u003e5\u003c/sup\u003e cells, respectively. Each treatment group comprised a minimum of five mice. Immunization (0.5 mg/kg mRNA-LNPs, intramuscular injection) was initiated upon the tumors reaching a size of 100 mm\u003csup\u003e3\u003c/sup\u003e or less. Tumor size was measured with calipers at 2\u0026ndash;3 days intervals until the tumor volume reached an endpoint of 1500 mm\u0026sup3;. The tumor volume was calculated using the following formula: The formula for calculation is as follows: length x width x width. The euthanasia of mice was conducted in accordance with the following criteria: a loss of \u0026ge;\u0026thinsp;20% of body weight, an inability to consume food, or the presence of tumors measuring\u0026thinsp;\u0026ge;\u0026thinsp;1.5 cm\u0026sup3;.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analysis\u003c/h3\u003e\n\u003cp\u003eBMDCs, spleens and lymph nodes were extracted from mice bearing tumors, and subsequently analyzed using flow cytometry to evaluate immune cell populations and their functional characteristics following various treatments. For further detail, please refer to the previously published article[14]. As for the blood samples, following the lysis of the blood then stained in a manner consistent with that of the spleen specimens. The staining of Ifn-γ, Tnf-α, and Gra B in tumor microenvironment Cd8\u003csup\u003e+\u003c/sup\u003e T cells necessitates a series of processing steps, including membrane permeabilisation and fixation.\u003c/p\u003e \u003cp\u003eSubsequent to staining, analysis of the cells was conducted via flow cytometry, thereby enabling the assessment of the frequency and activation status of discrete immune cell populations. The acquisition of data was conducted utilising a Beckman Coulter flow cytometer, and subsequent analyses were executed employing FlowJo or CytExpert software. The expression levels of surface markers were then quantified in order to evaluate the contribution of different immune cell populations to immune response.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of adenosine by LC-MS/MS\u003c/h2\u003e \u003cp\u003eThe tissue sample should be precisely weighed (0.1 g/0.5 g) and transferred into a 5-mL grinding tube. The tube should then be diluted to 0.5/1 mL, and the sample homogenised. Finally, 100 \u0026micro;L of tissue and serum should be taken. The addition of 100 \u0026micro;L of a 2.5% perchloric acid aqueous solution is required, followed by ultrasonication in order to precipitate proteins. The centrifuge should be operated at 12,000 rpm for a period of 20 minutes. Subsequently, 150 \u0026micro;L of the above mixture should be transferred and neutralised with 15 \u0026micro;L of a 1 mol/L NaOH aqueous solution. The mixture should be thoroughly agitated and subsequently filtered through a 0.22 \u0026micro;m membrane. The transfer of the sample to a brown sample vial is required for the subsequent instrumental analysis (Agilent HPLC 1200).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003escRNA-seq and data analysis for mouse tumors\u003c/h2\u003e \u003cp\u003eFor each scRNA-seq sample, subcutaneous tumors from three mice within the same group were pooled. Tumors were generated by subcutaneously injecting 2.5 \u0026times; 10⁵ RM1-Psma cells into 8-week-old C57BL/6 mice. After Cd73-LNPs treatments, the tumors collected from PBS groups and Cd73-LNPs were individually processed and combined into a single-cell suspension. Cd45\u003csup\u003e+\u003c/sup\u003e immune cells were isolated by staining the suspension with anti-mouse Cd45 for 30 min and sorted using a BD FACSAriaIII for downstream scRNA-seq analysis.\u003c/p\u003e \u003cp\u003eA total of 20 clusters were generated at a resolution of 0.6, and further identified into \u0026ldquo;Cd4 T\u0026rdquo;, \u0026ldquo;Cd8 T\u0026rdquo;, \u0026ldquo;NK\u0026rdquo;, \u0026ldquo;Macrophage\u0026rdquo;, \u0026ldquo;Monocyte\u0026rdquo;, \u0026ldquo;B cell\u0026rdquo;, \u0026ldquo;pDC\u0026rdquo;, \u0026ldquo;cDC1\u0026rdquo;, \u0026ldquo;cDC2\u0026rdquo;, \u0026ldquo;Neutrophil\u0026rdquo; and \u0026ldquo;unidentified\u0026rdquo; (not shown) using the expression of published markers. A detailed analysis of the steps involved, please refer to the references provided[15].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted with GraphPad Prism (Version 8.0, CA, USA). A p value less than 0.05 was considered statistically significant (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; ns, not significant).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSafety profile and in\u003c/b\u003e \u003cb\u003evivo\u003c/b\u003e \u003cb\u003eimmunogenicity of Cd73 protein induced by Cd73-LNPs.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe preparation of Cd73 mRNA vaccines (Cd73-LNPs) utilized lipid nanoparticles previously developed in our research, as depicted schematically in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The in vitro transcription of CD73 mRNA was evaluated using capillary electrophoresis, confirming that the mRNA met quality control standards, with no signs of degradation or residual impurities \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. We conducted a comprehensive characterization of the physical and chemical properties of the Cd73-LNPs. The average diameter of the Cd73-LNPs complex was determined to be 81.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 nm, with a polydispersity index (PDI) of 0.095\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cb\u003eTable\u0026nbsp;1)\u003c/b\u003e. Subsequently, we evaluated the translational efficiency of the Cd73-LNPs. Both western blot and flow cytometry analyses indicated that the transfection of BMDCs with Cd73-LNPs resulted in effective translation into Cd73 protein \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F, \u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e. The cytotoxicity of Cd73-LNPs in vitro was assessed using the CCK8 assay, which revealed that administration at a concentration of 10 \u0026micro;g/mL did not significantly affect the viability of BMDCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. Safety evaluations were conducted following the administration of Cd73-LNPs to ascertain that the mice exhibited a favorable tolerance to the therapy. Blood biochemical analyses revealed that key indicators of liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and creatine kinase (CK), remained within normal physiological ranges, suggesting the absence of hepatic or renal dysfunction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. Having established that Cd73-LNPs possess favorable characterization and safety profiles, the subsequent phase of the research involved assessing the immunogenicity of the Cd73 protein. In healthy C57BL/6 mice, following four intramuscular injections of Cd73-LNPs (5 \u0026micro;g per mouse), T cells from the spleen were co-cultured with BMDCs pretreated with Cd73-LNPs. Interferon-gamma (IFN-γ) production was quantified using an ELISpot assay \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e. The results demonstrated that BMDCs pretreated with Cd73-LNPs exhibited a significantly enhanced production of Ifn-γ compared to control BMDCs. This finding indicates that Cd73-LNPs effectively activated specific immune responses, thereby underscoring their potential as tumor-associated antigen mRNA vaccines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ-K\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe findings of this study illustrate the successful synthesis and delivery of Cd73 mRNA using lipid nanoparticles (LNPs), thereby verifying their effective expression within dendritic cells. Additionally, the research substantiates both the in vitro and in vivo safety profiles of Cd73-LNPs and demonstrates their capacity to activate immune responses, highlighted the potential of Cd73-LNPs as tumor-associated antigen mRNA vaccines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCd73-LNPs induced the maturation and activation of BMDCs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe efficient presentation of antigens by antigen-presenting cells (APCs) and the subsequent activation of adaptive immune cells are crucial for the efficacy of mRNA vaccines [16]\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. This study undertook a comprehensive investigation into the phenotypic modulation of BMDCs by Cd73-LNPs. Phenotypic alterations in BMDCs were assessed 24 hours post-transfection with Cd73-LNPs. Given that innate immune signaling often facilitates the expression of inflammatory cytokine genes to activate dendritic cells, we first evaluated cytokine gene expression in BMDCs using quantitative PCR (qPCR). The findings indicated that Cd73-LNPs treatment significantly induced the expression of proinflammatory cytokine genes Il-12p40, Ifn-α1, Ifn-α4, and Ifn-β1 in BMDCs, with a milder induction observed for Il-1β, Il-6, and Il-12α \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. ELISA results corroborated the upregulation of protein levels of Il-1β, Il-6, Il-12p70, and Ifn-β cytokines in the culture supernatant from cells treated with Cd73-LNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Consequently, it was determined that Cd73-LNPs induced the expression of proinflammatory cytokines in BMDCs. Furthermore, flow cytometry analysis revealed that Cd73-LNPs significantly upregulated the surface expression of costimulatory molecules, including Cd80, Cd86, H-2m, Cd40, Cd103, Ox40l, and 4-1bbl on BMDCs, indicating maturation and activation of these cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-H, \u003cb\u003eFigure S3)\u003c/b\u003e. Additionally, the expression of Ccr7 was upregulated \u003cb\u003e(Figure I, Figure S3)\u003c/b\u003e, which is essential for the directional homing of dendritic cells to lymphoid tissues[17].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings suggested that Cd73-LNPs can effectively enhance the expression of cytokines and costimulatory molecules in BMDCs, highlighting their potential to activate and mature dendritic cells for enhanced immunotherapeutic efficacy. Building upon these results, future studies will investigate the functional implications of these phenotypic changes in eliciting robust anti-tumor immune responses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCd73-LNPs activated potent humoral and cellular immunity in\u003c/b\u003e \u003cb\u003evivo\u003c/b\u003e.\u003c/p\u003e \u003cp\u003emRNA vaccines are recognized for their ability to induce robust immune responses by activating both humoral and cellular immunity[2]. The roles of Cd73-LNPs in the in \u003cem\u003evivo\u003c/em\u003e immune response were investigated based on the phenotypic modulation of BMDCs by Cd73-LNPs. In tumor-free C57BL/6 mice models, after two rounds of immunization, serum samples and T cells isolated from spleen were collected for functional assessment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). CD73 serves as the rate-limiting enzyme in adenosine metabolism, however, our findings indicated that Cd73-LNPs did not significantly alter serum adenosine levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Notably, a decrease in serum Cd73 protein concentrations was observed, accompanied by an increase in Cd73-specific immunoglobulin G (IgG) concentrations \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, suggesting that Cd73-LNPs effectively elicited a humoral immune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs the largest secondary lymphoid organ, the spleen serves as a critical site for the initiation of immune responses by mRNA vaccines[18, 19]. Our observations indicate that Cd73-LNPs significantly increased the proportion of Cd3\u003csup\u003e+\u003c/sup\u003e T cells in the spleen, with a particular emphasis on Cd8\u003csup\u003e+\u003c/sup\u003e T cells. In contrast, there was a notable decrease in the proportion of Cd11\u003csup\u003e+\u003c/sup\u003e dendritic cells; however, their surface expression of Cd80 and Cd86 was elevated. Additionally, the proportion of F4/80\u003csup\u003e+\u003c/sup\u003e/Cd206\u003csup\u003e+\u003c/sup\u003e macrophages was diminished, suggesting a transformation of these macrophages. No significant changes were observed in the proportions of natural killer (NK) cells and B cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D, \u003cb\u003eFigure S4-5)\u003c/b\u003e. Given that Cd73-LNPs were associated with increased serum concentrations of specific IgG, we further evaluated the proportion of activated plasma cells in the spleen. The results revealed a higher proportion of B220/Cd138-positive plasma cells, indicating that Cd73-LNPs facilitate B cell activation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cb\u003eFigure S6)\u003c/b\u003e. Furthermore, we found that Cd73-LNPs promote the activation of Cd4\u003csup\u003e+\u003c/sup\u003e and Cd8\u003csup\u003e+\u003c/sup\u003e T cells in vitro \u003cb\u003e(Figure S7)\u003c/b\u003e and enhance the secretion of effector molecules such as Ifn-γ, Granzyme B, and Tnf-α by Cd8\u003csup\u003e+\u003c/sup\u003e T cells in vivo \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H, \u003cb\u003eFigure S8)\u003c/b\u003e. Moreover, the transcription factor Eomes, known for its role in the activation of Cd8\u003csup\u003e+\u003c/sup\u003e T cells[20], was observed to have increased expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, \u003cb\u003eFigures S8-9\u003c/b\u003e). These results indicate that Cd73-LNPs can effectively enhance both humoral and cellular immune responses in vivo without affecting serum adenosine concentrations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCd73-LNPs improved immune cell infiltration in the tumor microenvironment and enhanced anti-tumor efficacy\u003c/h2\u003e \u003cp\u003eIt has been demonstrated that Cd73-LNPs possess favorable safety profiles and can effectively activate both humoral and cellular immune responses in vivo. We next examined the anti-tumor efficacy of Cd73-LNPs in subcutaneous tumor models, including B16-Ova (melanoma), TC-1 (cervical cancer), ID8 (ovarian cancer) and RM1-Psma (prostate cancer). E7 is a specific mRNA vaccine therapeutic target for the TC-1 models[21], and prostate specific membrane antigen (PSMA) is considered an effective target for prostate cancer treatment, has been validated in CAR-T, and mRNA vaccines[22]. In this study, following administration of the four Cd73-LNPs, a significant advantage in both tumor volume reduction and overall survival in the aforementioned models \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-I, \u003cb\u003eFigure S10\u003c/b\u003e). Tumor volumes consistently remained below 500 mm\u0026sup3; after Cd73-LNPs treatments, demonstrating superior antitumor ability of Cd73-LNPs. The complete response rates for B16-Ova model, TC-1 model and ID8 model were 40% (2/5), 60% (3/5) and 100% (5/5), respectively. In the RM1-Psma model, Cd73-LNPs demonstrated significantly superior efficacy compared to Psma-LNPs, in the TC-1 model, the efficacy of Cd73-LNPs was comparable to that of the mRNA vaccine targeting the specific antigen E7.\u003c/p\u003e \u003cp\u003eProstate cancer is characterized as an immunologically cold tumor, marked by a tumor microenvironment deficient in effective immune cell infiltration[23]. Surprisingly, Cd73-LNPs have been observed to enhance immune cell infiltration in the prostate cancer tumor microenvironment, including Cd45-positive immune cells, Cd8\u003csup\u003e+\u003c/sup\u003e T cells, and NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, \u003cb\u003eFigure S11\u003c/b\u003e). In addition, Cd73-LNP have been shown to facilitate the secretion of Ifn-γ and Gra B by Cd8\u003csup\u003e+\u003c/sup\u003e T cells within the tumor microenvironment in prostate cancer model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-L, \u003cb\u003eFigure S12\u003c/b\u003e). Since Cd73-LNPs were found to achieve 100% complete remission in the ID8 model, we investigated the proportion of memory T cells present in the blood of ID8 model mice. The results showed that Cd73-LNPs had significantly increased the proportion of Cd44/Cd62l-positive Cd8\u003csup\u003e+\u003c/sup\u003e T cells (\u003cb\u003eFigure S13\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThese results indicate that Cd73-LNPs enhance Cd8\u003csup\u003e+\u003c/sup\u003e T cell infiltration into the tumor microenvironment, effectively suppress tumor growth, and demonstrate promising antitumor efficacy across multiple tumor models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCd73-LNPs activated Cxcl/Cxcr signaling to promote Cd8\u003csup\u003e+\u003c/sup\u003e T cell infiltration\u003c/h2\u003e \u003cp\u003eActivation and recruitment of CD8\u003csup\u003e+\u003c/sup\u003e T cells to the tumor microenvironment are essential for effective anti-tumor responses. In our study utilizing the RM1-Psma model, we demonstrated that Cd73-LNPs enhanced immune cell infiltration into the tumor microenvironment and stimulated the secretion of effector molecules by Cd8\u003csup\u003e+\u003c/sup\u003e T cells. Nonetheless, the precise mechanism by which Cd73-LNPs facilitated the recruitment of Cd8\u003csup\u003e+\u003c/sup\u003e T cells to the tumor microenvironment remained to be elucidated. To gain a deeper understanding of the impact of Cd73-LNPs on T cells within the tumor immune microenvironment, we conducted single-cell RNA sequencing (scRNA-seq) on Cd45\u003csup\u003e+\u003c/sup\u003e immune cells isolated from RM1-Psma xenografts harvested from C57BL/6J mice treated with either PBS or CD73-LNPs. Following quality control and filtering, we successfully captured a total of 12,327 cells, obtained their single-cell transcriptomes, and identified 21 transcriptionally homogeneous clusters using the t-distributed stochastic neighbor embedding (t-SNE) method \u003cb\u003e(Figure S14A)\u003c/b\u003e. These clusters were annotated into 11 distinct cell types based on their transcriptional profiles and the SingleR package[15], including Cd3\u003csup\u003e+\u003c/sup\u003e T cells, Cd4\u003csup\u003e+\u003c/sup\u003e T cells, Cd8\u003csup\u003e+\u003c/sup\u003e T cells, natural killer cells (NK), macrophages, monocytes, B cells, plasmacytoid dendritic cells (pDC), conventional dendritic cell 1 (cDC1), cDC2 and neutrophils. Notably, the proportion of Cd8\u003csup\u003e+\u003c/sup\u003e T cells exhibited the most significant increase. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003eIn our study, we employed t-SNE analysis to categorize Cd8\u003csup\u003e+\u003c/sup\u003e T cells into distinct subsets, including Cd8\u003csup\u003e+\u003c/sup\u003e effector T cells, exhausted Cd8\u003csup\u003e+\u003c/sup\u003e T cells, na\u0026iuml;ve Cd8\u003csup\u003e+\u003c/sup\u003e T cells, early activated T cells, and tissue-resident memory Cd8\u003csup\u003e+\u003c/sup\u003e T cells \u003cb\u003e(Figure S14B-C)\u003c/b\u003e. Compared to mice treated with PBS, those treated with Cd73-LNPs exhibited an increased population of effector Cd8\u003csup\u003e+\u003c/sup\u003e T cells and a decreased population of exhausted Cd8\u003csup\u003e+\u003c/sup\u003e T cells \u003cb\u003e(Figure S14A)\u003c/b\u003e. Additionally, volcano plots illustrating differentially expressed genes in Cd8\u003csup\u003e+\u003c/sup\u003e T cells between the two groups corroborated the upregulation of genes associated with Cd8\u003csup\u003e+\u003c/sup\u003e effector T cells, such as Gzma, Gzmb, and Gzmk \u003cb\u003e(Figure S14D)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAn analysis of intercellular communication networks revealed that Cxcl-related signaling was significantly upregulated in mice treated with Cd73-LNPs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, S14E\u003cb\u003e)\u003c/b\u003e. The kinetics of chemokine expression within the tumor microenvironment (TME), along with alterations in chemokine receptor expression on immune cells, are likely determinants of the immune cell composition within the TME [24]. Subsequently, we conducted KEGG and GO enrichment analyses on the upregulated genes in intratumoral Cd8\u003csup\u003e+\u003c/sup\u003e T cells. The results indicated enrichment in pathways related to lymphocyte activation and migration, chemokine signaling, and cytokine-cytokine receptor interactions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E\u003cb\u003e)\u003c/b\u003e. Chemokines facilitate directed cell migration by binding to chemokine receptors (CXCR). Consequently, we employed flow cytometry to assess the expression of CXCR receptors, which facilitate the migration of Cd8\u003csup\u003e+\u003c/sup\u003e T cells in the blood, draining lymph nodes (DLN), and spleen. The findings demonstrated that post-Cd73-LNPs treatment, Cxcr3 expression was upregulated on Cd8\u003csup\u003e+\u003c/sup\u003e T cells from the blood, DLN, and spleen; Cx3cr1 expression was upregulated on Cd8\u003csup\u003e+\u003c/sup\u003e T cells from the blood and spleen; and Cxcr6 and Ccr5 expressions were upregulated exclusively in blood Cd8\u003csup\u003e+\u003c/sup\u003e T cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-I, \u003cb\u003eFigure S15-16)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe findings presented herein substantiate that Cd73-LNPs augment the migratory capacity of Cd8\u003csup\u003e+\u003c/sup\u003e T cells through the activation of the Cxcl/Cxcr signaling pathway. Furthermore, Cd73-LNPs have been demonstrated to decrease the infiltration of exhausted T cells within the tumor microenvironment, underscoring their considerable therapeutic potential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCd73-LNPs activated BMDCs through Tlr3 signaling\u003c/h2\u003e \u003cp\u003eToll-like receptors (TLRs) initiate both shared and distinct signaling pathways that modulate immune responses[25]. Notably, among all TLRs, only the endosomal TLR3 does not trigger systemic inflammation and facilitates the cross-priming of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells by dendritic cells[26, 27]. TLR3 activation leads to downstream signaling cascades involving NF-kB, MAPKs, and IRF3, which result in the expression of inflammatory mediators and cytokines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe collected BMDCs which treated with Cd73-LNPs for 24 hours, transcriptome analysis was then performed via RNA sequencing. Subsequent transcriptome analysis was conducted using RNA sequencing. Principal component analysis (PCA) demonstrated robust replication within clusters and revealed significant differences between the Cd73-LNPs treatment group and PBS control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The volcano plot shows that the expression of the genes associated with BMDC activation is significantly increased, including Ms4a6b, Slfn4, Fcgr1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The Gene Set Enrichment Analysis (GSEA) revealed a significant enrichment of the Toll-like receptor signaling pathway, specifically the TLR3 and TLR4 signaling pathways \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Quantitative PCR (qPCR) experiments further confirmed that among the Toll-like receptors in mice, Tlr3 exhibited the most pronounced upregulation in expression following treatment with Cd73-LNPs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. To ascertain whether the activation of BMDCs by Cd73-LNPs was contingent upon the Tlr3 signaling pathway, BMDCs were pretreated with small molecule inhibitors targeting Tlr3. Subsequently, the expression of cytokine genes and costimulatory molecules in BMDCs treated with Cd73-LNPs was analyzed. The findings demonstrated that CU CPT4a, a Tlr3-specific inhibitor, effectively inhibited the Cd73-LNPs-induced production of cytokines (Il-1β, Il-6, Ifn-α4, and Ifn-β1) and costimulatory molecules (Cd86, H-2m) in BMDCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H, \u003cb\u003eFigure S17).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSubsequently, we collected BMDCs from Tlr3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and observed that treatment with Cd73-LNPs significantly increased the phosphorylation levels of protein kinases, including NF-κB, mitogen-activated protein kinases (MAPKs), and Irf3, in wild-type (WT) BMDCs. However, this phosphorylation was substantially diminished in Tlr3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e BMDCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-J\u003cb\u003e)\u003c/b\u003e. Additionally, we assessed the therapeutic efficacy of Cd73-LNPs in subcutaneous tumor models of TC-1 and RM1-Psma using Tlr3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The tumor inhibitory effect of Cd73-LNPs was nearly completely nullified in Tlr3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK-N, \u003cb\u003eFigure S18)\u003c/b\u003e. Furthermore, we conducted a comparative analysis of immune cell populations and proportions in the blood of wild-type and Tlr3 knockout mice using flow cytometry, which revealed no significant differences between the two groups \u003cb\u003e(Figure S19)\u003c/b\u003e. These findings underscore the critical role of Tlr3 in mediating the antitumor effects induced by Cd73-LNPs. Collectively, the data strongly indicate that the antitumor efficacy of Cd73-LNPs necessitates both effective antigen expression and the activation of Tlr3 signaling pathways.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the potential of Cd73 as a target for mRNA vaccines was investigated, with findings indicating that Cd73 mRNA vaccines effectively inhibit tumor growth in murine models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Mechanistically, the function of Cd73-LNPs was shown to depend on Tlr3 signaling in dendritic cells, leading to the upregulation of chemokine receptor expression on Cd8\u003csup\u003e+\u003c/sup\u003e T cells. Furthermore, a comparative analysis was performed to evaluate the therapeutic efficacy of Cd73-LNPs in combination with a Cd73 monoclonal antibody (anti-Cd73) and the Cd73 inhibitor APCP in a prostate cancer model. The results revealed that APCP exhibited the greatest ability to inhibit adenosine metabolism in both serum and the tumor microenvironment. However, Cd73-LNPs were found to exert the most potent tumor-suppressive effect \u003cb\u003e(Figure S20A-G\u003c/b\u003e). These findings underscore the feasibility of utilizing Cd73 mRNA vaccines for the treatment of the aforementioned cancers, highlighting a promising strategy for cancer immunotherapy.\u003c/p\u003e \u003cp\u003eThe efficacy of mRNA vaccine approaches demonstrates notable variability, as most exhibit limitations related to the immunogenicity of the antigenic targets, the lack of systemic immune responses, and an inability to induce the rapid and adaptable immune responses that are likely essential for the effective suppression of rapidly evolving cancers[2]. Tumor-associated antigens have been demonstrated to exhibit innate immune tolerance, a factor which contributes to their insufficient immunogenicity[28, 29]. However, Cd73, as a tumor-associated antigen, has been shown to elicit potent anti-tumor immune responses. In the TC-1 model, the efficacy of the Cd73-LNPs was comparable to that of the TC-1-specific target mRNA vaccine E7-LNPs, with a CR of 60% (3/5). In the RM1-Psma model, Cd73-LNPs demonstrated significantly superior efficacy to Psma-LNPs, despite PSMA being considered a prostate cancer-specific target. The results of scRNA-seq indicated a significant increase in the proportion of effector T cells within the tumor microenvironment, coupled with a notable reduction in the proportion of exhausted T cells. The therapeutic efficacy of Cd73-LNPs is likely due to its dual mechanisms of activating anti-tumor immunity and mitigating the immunosuppressive state within the tumor microenvironment.\u003c/p\u003e \u003cp\u003eFollowing administration of Cd73-LNPs, a notable enhancement in chemokine signaling and the infiltration of effector T cells within the tumor microenvironment was observed. Enrichment analysis further identified a significant enrichment of pathways associated with the activation and migration of Cd8\u003csup\u003e+\u003c/sup\u003e T cells. Chemokines have been documented to facilitate the recruitment of circulating CD8\u003csup\u003e+\u003c/sup\u003e T cells into the tumor microenvironment through interaction with their specific receptors[30]. The following chemokine receptors and their corresponding ligands are recognized as key mediators in the migration and activation of effector T cells: CXCR3 (CXCL9, CXCL10), CXCR1 (CXCL1), CXCR6 (CXCL16), and CCR5 (CCL5)[31]. The results of this study demonstrated an upregulation of all aforementioned receptors on the surface of Cd8\u003csup\u003e+\u003c/sup\u003e T cells in the bloodstream, with a concurrent upregulation of Cxcr3 expression on Cd8\u003csup\u003e+\u003c/sup\u003e T cells from the spleen and draining lymph nodes (DLN). Although the CXCR3/CXCL9-CXCL10 axis is also involved in the migration of regulatory T cells (Tregs)[32], in this study, it predominantly facilitated the migration of effector T cells.\u003c/p\u003e \u003cp\u003eMoreover, this study demonstrated that Cd73-LNPs activate TLR3 signaling in dendritic cells, thereby enhancing antitumor immunity. Unlike most vaccines, which require adjuvants to boost immunogenicity, mRNA inherently possesses immunogenic properties and acts as a pathogen-associated molecular pattern. It exhibits affinity for various TLRs, such as TLR3, TLR7, and TLR8, as well as intracellular sensors[3, 33]. These characteristics facilitate the swift initiation of innate immune responses, in conjunction with the activation of adaptive immunity, as the mRNA payload is translated into full-length proteins and subsequently processed as antigens[3]. Conversely, lipid nanoparticles delivery systems have the potential to activate innate immune-related signaling pathways. For instance, the cationic lipids developed by Xia et al. have been demonstrated to activate Toll-like receptor 4. (TLR4) signaling in dendritic cells, thereby enhancing anti-tumor immunity[34]. Similarly, the lipids synthesized by Anderson et al. have been reported to activate the stimulator of interferon genes (STING) signaling pathway, thus augmenting immune responses in vivo[35]. The mRNA delivery system utilized in this study consisted of LNPs previously engineered to induce cytokine secretion in bone marrow-derived dendritic cells (BMDCs), although the precise mechanism remains to be elucidated. In this investigation, Luc-LNPs were observed to induce a moderate upregulation of Tlr3 expression in BMDCs, whereas Cd73-LNPs demonstrated a more substantial upregulation of Tlr3. This suggests that the activation of Tlr3 by Cd73-LNPs likely represents a synergistic interaction between the delivery system and Cd73 mRNA.\u003c/p\u003e \u003cp\u003eThis study is limited by the lack of investigation into the specific mechanism underlying the ineffectiveness of Cd73-LNPs treatment following Tlr3 deficiency. Furthermore, Cd8\u003csup\u003e+\u003c/sup\u003e T cell chemokine receptor expression was only validated in the RM1 model following Cd73-LNPs treatment. Moreover, the therapeutic efficacy of Cd73-LNPs was not explored further in humanized mice or other models.\u003c/p\u003e \u003cp\u003eIn conclusion, we have demonstrated that the Cd73 protein is highly immunogenic, the Cd73 mRNA vaccine has been shown to safely and effectively induce antitumor immunity, suggesting significant potential for clinical translation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLNPs: Lipid nanoparticles, NK: Natural killer, BMDCs:\u003c/p\u003e\n\u003cp\u003eBone marrow-derived dendritic cells, PDI: Polydispersity index, ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, BUN: Blood urea nitrogen, CREAT: creatinine. Gra B: granzyme B, TLR: toll-like receptor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e The Institutional Animal Care and Use Committee (IACUC) at Sun Yat-Sen University has formally approved all the animal procedures (SYSU-IACUC-2025-002827).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eAll authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u0026nbsp;\u003c/strong\u003eAll data are included in the paper or the supplemental information. Additional data are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by Shenzhen Fundamental Research Program (Grant No. JCYJ20240813150249044, JCYJ20250604143756074), Guangdong Basic and Applied Basic Research Foundation (Grant No. 2023A1515030058), the National Natural Science Foundation of China (Grant No. 82272689).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions:\u0026nbsp;\u003c/strong\u003eYuandong Xu, Fei Cao, Ze-Xiu Xiao, Gao-feng Zha, and Jun Pang. developed this concept and designed this study. Yuandong Xu, Fei Cao, Haowei Qiu, Yiting Zhang, Zhen Xu, Yunru He performed the experiments and acquired the data. Yuandong Xu and Jun Pang acquired the funding. Yuandong Xu, Fei Cao, Haowei Qiu, and Yi Zhang performed data analysis. Yuandong Xu and Yi Zhang performed single-cell transcriptome sequencing and RNA sequencing analysis. Yuandong Xu, Ze Xiu Xiao, Gao-feng Zha, and Jun Pang edited and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e It is imperative to express our profound gratitude to the dedicated staff at Shenzhen MagicRNA Biotech for their invaluable support during the implementation of this study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLorentzen CL, Haanen JB, Met \u0026Ouml;, Svane IM. Clinical advances and ongoing trials on mRNA vaccines for cancer treatment. Lancet Oncol. 2022; 23: e450-e8.\u003c/li\u003e\n \u003cli\u003eYaremenko AV, Khan MM, Zhen X, Tang Y, Tao W. Clinical advances of mRNA vaccines for cancer immunotherapy. Med. 2025; 6: 100562.\u003c/li\u003e\n \u003cli\u003eLi H, Min L, Du H, Wei X, Tong A. 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Chemokines and the immune response to cancer. Immunity. 2021; 54: 859-74.\u003c/li\u003e\n \u003cli\u003eMoreno Ayala MA, Campbell TF, Zhang C, Dahan N, Bockman A, Prakash V, et al. CXCR3 expression in regulatory T cells drives interactions with type I dendritic cells in tumors to restrict CD8(+) T cell antitumor immunity. Immunity. 2023; 56: 1613-30.e5.\u003c/li\u003e\n \u003cli\u003eCao LL, Kagan JC. Targeting innate immune pathways for cancer immunotherapy. Immunity. 2023; 56: 2206-17.\u003c/li\u003e\n \u003cli\u003eZhang H, You X, Wang X, Cui L, Wang Z, Xu F, et al. Delivery of mRNA vaccine with a lipid-like material potentiates antitumor efficacy through Toll-like receptor 4 signaling. Proc Natl Acad Sci U S A. 2021; 118.\u003c/li\u003e\n \u003cli\u003eMiao L, Li L, Huang Y, Delcassian D, Chahal J, Han J, et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat Biotechnol. 2019; 37: 1174-85.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cancer mRNA vaccines, Cd73, Tlr3, Cd8+ T cells, Immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-8715323/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8715323/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003emRNA vaccines have emerged as highly promising therapeutic agents in the field of cancer immunotherapy. Nevertheless, the current limited immunogenicity of target antigens, along with a lack of systemic immune response, poses significant challenges to the effective implementation of mRNA vaccines. This study sought to evaluate the immunogenicity of the Cd73 protein and elucidate the mechanisms through which the Cd73 mRNA vaccines mediated its antitumor immune effects in murine models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eIn vitro transcription synthesis of Cd73 mRNA was employed to prepare a Cd73 mRNA vaccine based on lipid nanoparticles (Cd73-LNPs) using a microfluidic device. The immunogenicity of Cd73 was assessed via ELISPOT assays. The capability of Cd73-LNPs to activate anti-tumor immune responses was investigated via flow cytometry. The therapeutic efficacy of Cd73-LNPs was tested in models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Furthermore, the mechanism of action of Cd73-LNPs was elucidated through single-cell transcriptomics sequencing and RNA transcriptomics sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe Cd73 protein exhibited potent immunogenicity. In vitro, Cd73-LNPs have been observed to promote the maturation and activation of primary dendritic cells. In vivo, they have been observed to activate both humoral and cellular immune responses, stimulating secretion of Ifn-γ and granzyme B, effectively suppressing tumor growth in models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Mechanistically, Cd73-LNPs activate dendritic cells through toll-like receptor 3 signaling, enhancing their activation and upregulating chemokine receptor expression on Cd8\u003csup\u003e+\u003c/sup\u003e T cells, which promotes Cd8\u003csup\u003e+ \u003c/sup\u003eT cell infiltration into the tumor microenvironment. Safety evaluations revealed that Cd73-LNPs do not produce toxic side effects on vital organs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Cd73 mRNA vaccines have been demonstrated to safely and effectively induce antitumor immunity through toll-like receptor 3 signaling, indicating considerable potential for clinical application.\u003c/p\u003e","manuscriptTitle":"Cd73-LNPs promotes antitumor T-cell immunity via amplifying Tlr3-mediated immunostimulatory dendritic cell activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 09:08:07","doi":"10.21203/rs.3.rs-8715323/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82b51201-71e1-4422-8bd4-6d1db0133a09","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T17:08:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 09:08:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8715323","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8715323","identity":"rs-8715323","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}