CircRNA-loaded DC vaccine in combination with low-dose gemcitabine induced potent anti-tumor immunity in pancreatic cancer model

doi:10.21203/rs.3.rs-5034407/v1

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CircRNA-loaded DC vaccine in combination with low-dose gemcitabine induced potent anti-tumor immunity in pancreatic cancer model

https://doi.org/10.21203/rs.3.rs-5034407/v1

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Although promising, dendritic cell (DC) vaccines may not suffice to fully inhibit tumor progression alone, mainly due to the short expression time of the antigen in DC vaccines, immunosuppressive tumor microenvironment, and tumor antigenic modulation. Overcoming the limitations of DC vaccines is expected to further enhance their anti-tumor effects. In this study, we constructed a circRNA-loaded DC vaccine utilizing the inherent stability of circular RNA to enhance the expression level and duration of the antigen within the DC vaccine. Meanwhile we combined it with gemcitabine and validated their therapeutic efficacy in the Panc02 tumor model. We found that the use of DC vaccine alone can reach a tumor inhibition rate of 69%, and the effect was further enhanced when combined with gemcitabine, reaching a tumor inhibition rate of 89%. The combined treatment achieved a synergistic effect, which not only reduced immunosuppressive Tregs but also induced immunogenic cell death, leading to antigen spreading and reducing immune evasion caused by tumor antigenic modulation. As a result, the survival of the mice was significantly prolonged. Our research provides a promising approach for the clinical treatment of pancreatic cancer.

Immunotherapy

DC vaccine

Circular RNA

Gemcitabine

Immunogenic cell death

Cancer immunotherapy has attracted considerable interest due to its ability to persistently inhibit tumor progression, and exhibit fewer adverse effects[1]. Dendritic cells (DC) are considered the most effective antigen-presenting cells, playing a key role in both innate and adaptive immunity. They uptake, and present antigens, activate naïve T cells, induce a strong antigen-specific T cell response, and generate immune memory[2]. As a result of these attributes, DCs have emerged as a promising tool for cancer treatment[3]. In contrast to conventional cancer vaccines, DC vaccines possess the capability to directly activate T cells, as the antigens are expressed endogenously. This mechanism leads to a substantial enhancement of the antigen-specific T cell response[4]. There are many methods for loading tumor antigens in DC vaccines, within which circular RNA (circRNA) is very promising[5]. Compared to linear mRNA, circular RNA has higher stability, which prolongs the duration of protein translation and increases protein yield[6, 7]. At the same time, unmodified circular RNA has fewer side effects, exhibiting lower immunogenicity and cytotoxicity[8, 9]. In recent years, an increasing number of DC vaccines have demonstrated their ability to enhance patient survival and promote positive responses in clinical trials[10, 11].

The DC vaccines, although they can induce antigen-specific T cell responses more directly compared to other immunotherapies, still have limited efficacy. This is mainly due to the immunosuppressive tumor microenvironment (TME) and the tumor antigenic modulation[2, 12]. Overcoming the limitations of DC vaccines by combining them with other therapies is expected to further enhance anti-tumor effects. Gemcitabine (Gem), recognized as the standard therapeutic agent for individuals diagnosed with advanced pancreatic cancer, is insufficient in fully halting tumor progression when administered as a monotherapy[13]. Previous research indicated a strong correlation between an increased presence of intratumoral CD8⁺ T cells and extended survival rates among pancreatic cancer patients[14]. Tumor cells employ various escape mechanisms that render them insensitivity to cytotoxic T lymphocyte (CTL) responses, whereas Gem has been shown to enhance the susceptibility of tumor cells to CTL-mediated responses[15]. Furthermore, previous studies indicated that Gem selectively diminished the population of immunosuppressive cells within TME, including myeloid-derived suppressor cells (MDSC)[16] and regulatory T cells (Treg)[17]. Concurrently, it appeared to facilitate the uptake of antigens from apoptotic tumor cells by local DCs[18]. Consequently, the combination of Gem with DC vaccines may yield a synergistic therapeutic effect.

FAPα and survivin are considered two promising tumor-associated antigens (TAA). In our previous research, we developed a DNA vaccine called sPD1/FS that combines human sPD1, FAPα, and survivin. This vaccine was designed to target DCs via sPD1 and stimulated the generation of antigen-specific T cells against cancer-associated fibroblasts (CAF) and tumor cells[19]. However, its anti-tumor effect was limited. Here, we have developed a new DC vaccine that loads the antigens FAPα and survivin using circular RNA to induce a stronger CD8⁺ T cell response and compared it to the sPD1/FS vaccine. We further combined the DC vaccine with Gem to address the limitation of our DC vaccine. The combination treatment significantly extended the survival of mice, underscoring its potential as a robust therapeutic strategy in the fight against cancer.

2.1 Cell lines, mice, and tumor model

The murine Panc02 cell lines were provided by National Center for Nanoscience and Technology (Beijing, China). Female C57BL/6 mice (6–8 weeks old) were purchased from Changsheng Biotechnology Co., Ltd (Liaoning, China). A total of 3×10⁵ murine Panc02 tumor cells were injected subcutaneously into the right lower flank of mice. Mouse body weights and tumor sizes were measured using an electronic scale and a Vernier caliper every two days, respectively. Tumor size was calculated by the formula: V= (length×width²)/2 (mm³). The mice were euthanized for the following ethical reasons, including tumor ulceration, tumors growing to 2000 mm³, or a decline in health (such as loss of appetite or significant weight loss).

2.2 Sources of plasmids

The sPD1/FS plasmids (expressing the human sPD1, FAPα, and survivin) were constructed following previously described[19]. The pET9a-FS plasmid (carrying the circular RNA scaffold and antigen FS) was synthesized by the Jinsrui Biotechnology Co., Ltd (Nanjing, China).

2.3 Preparation and Purification of Circular RNA

Using the linearized pET9a-FS plasmid as a template, circular RNA precursors were generated through in vitro transcription utilizing a T7 transcription kit (NEB, USA). Heat the RNA precursor with an appropriate amount of DNase I (NEB, USA) at 37°C for 15 minutes. Purify the resulting product using the Monarch RNA purification kit. Next, add 2 mM GTP (thermofisher, USA) and mix it with the column-purified product. Heat the mixture at 55°C for 15 minutes. Treat the mixture with RNase R (Novoprotein, China), heat it at 37°C for 15 minutes, and perform column purification to obtain circular RNA.

2.4 Generation of BMDCs in vitro

Bone marrow cells were isolated from the femur and tibia of naïve mice. Cells were plated in culture dishes, and 20 ng/ml of GM-CSF (Absin, China) was added. The cells were cultured at 37°C with 5% CO₂. On the 3rd day, an equal volume of complete medium was added to the culture dishes along with additional GM-CSF. On the 6th and 8th days, half of the medium was replaced. On the 10th day, the cells were collected, and were resuspended and transferred back into the new culture dish, adding GM-CSF (10 ng/ml) and LPS (1 µg/ml) (Sigma, USA). After continuing the culture for 24 hours, mature BMDCs were obtained.

2.5 Construction of DC vaccine

After heating circular RNA at 70°C for 3 minutes, cool it on ice for an additional 3 minutes. Next, the RNA was mixed with DOTAP (Santa Cruz, USA) at a ratio of 1:2 in serum-free medium at room temperature for 20 minutes. Then, 2×10⁶/ml BMDCs were added and incubated at 37°C for 4 hours. The cells were transferred to medium supplemented with the previously indicated concentration of GM-CSF for a 24-hour incubation.

2.6 In vivo immune strategies

The sPD1/FS vaccine was administered via intramuscular injection in hind limbs, with 100µg on days 0, 2, and 5. The DC vaccine was administered via tail vein injection in mice, with 1×10⁶ DCs injected each time, once every 7 days, for a total of 2 injections. Gemcitabine (Hisun, China) was administered via intraperitoneal injection, with 15 mg/kg, once every 3 days, for a total of 4 injections.

2.7 IFN-γ ELISpot assay

The BD Biosciences ELISpot kit was utilized for the experiment, following the procedures from the manufacturer.

2.8 Cell staining and flow cytometry

For staining mouse spleen cells, the spleen was removed from the mice, ground, and the red blood cells were lysed to prepare a single-cell suspension for extracellular staining. After surface staining, cell fixation and permeabilization were performed, followed by intracellular staining. The staining of tumor cells was digested with liberase (Roche, Switzerland) and DNase I (Sigma, USA), while the remaining procedures followed the same protocol as for spleen cells. All antibodies utilized in the staining process were sourced from BioLegend.

2.9 RNAseq

The integrity and purity of RNA extracted from tumor tissues (n = 3) were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Santa Clara, USA) and the NanoPhotometer® spectrophotometer (IMPLEN, USA). Sequencing libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA). The raw data obtained were first processed using fastp software to obtain clean reads. Use HISAT2 to build an index of the reference genome and align paired-end clean reads to the reference genome. Use featureCounts to calculate gene expression levels. Perform differential expression analysis between two comparison groups using DESeq2 software. Conduct differential gene enrichment analysis through clusterProfiler.

2.10 Statistical analysis

All experiments were performed at least two times. All statistical analyses were conducted using GraphPad Prism. Comparisons between the two groups were examined utilizing an unpaired T-test or two-way ANOVA, and the outcomes were presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance.

3.1 Construction of circRNA-loaded DC vaccine

We designed a circular RNA loading the antigens FAPα and survivin (circRNA^FS) by a permuted intron-exon system and successfully synthesized it in vitro (Fig. 1A-B). Following, we isolated bone marrow cells from naïve C57BL/6 mice femurs and tibias, stimulated and cultured them in vitro, and successfully obtained DCs (Fig. 1C). We transfected the circRNA^FS into DCs, and confirmed its expression by Western blot (Fig. 1D). The transfection efficiency was approximately 74.44% (Fig. 1E). Altogether, we successfully constructed a circRNA-loaded DC vaccine.

3.2 Immunogenicity of circRNA-loaded DC vaccine

Next, we evaluated the immunogenicity of circRNA-loaded DC vaccine (DC^FS) in vivo and compared it with the sPD1/FS vaccine (Fig. 2A). We found that compared to the sPD1/FS group, the number of CD8⁺ T cells in the DC^FS group significantly increased, while CD4⁺ T cells did not show any significant changes (Fig. 2B). It is worth noting that we found that the antigen-specific T cell response in both the DC group and the DC^FS group was significantly enhanced whether circular RNA was loaded or not. Adding 1×10⁶ splenocytes cannot compare the differences between the two groups. After diluting the number of splenocytes to 3×10⁵, the antigen-specific T cell response in the DC^FS group is significantly stronger than that in the DC group (Fig. 2C). Subsequently, we evaluated the antibody levels induced by the vaccine. However, the results showed that the levels of both survivin and FAPα antibodies were stronger in the sPD1/FS group compared to the DC^FS group (Fig. 2D). This indicated that the immune response induced by the DC^FS group was more skewed towards the Th1 type compared to the sPD1/FS group.

3.3 circRNA-loaded DC vaccination had a better therapeutic effect on Panc02 pancreatic cancer

We evaluated the anti-tumor effect of the DC^FS vaccine in an established murine Panc02 model. The results showed that the DC^FS group exhibited a more significant therapeutic effect than the other groups, achieving a tumor inhibition rate of 69% (Fig. 3A). In the spleen, we observed an increase in the number of CD8⁺ T cells and CD4⁺ T cells in the DC^FS group, along with an enhanced antigen-specific T cell response (Fig. 3B-C, Supplementary Fig. 1A). Similarly, we found that the DC^FS group showed a significant increase in the number of intratumoral CD45⁺ immune cells, CD8⁺ T cells, CD4⁺ T cells and NK cells (Fig. 3D-F). The MDSCs in the DC^FS group were significantly decreased, while Tregs were significantly increased. (Fig. 3G-H). We also detected the levels of Th1-type cytokines within the tumor. We found that the IFN-γ levels of the DC^FS group did not increase significantly as in the ELISpot results; they only slightly increased, with no significant difference, and the same went for IL-2 (Supplementary Fig. 1B). Altogether, the DC^FS vaccine had a better therapeutic effect on Panc02 pancreatic cancer, but it led to an increase in Tregs.

3.4 Combining low-dose Gem further enhanced the anti-tumor effect.

Low-dose Gem can selectively deplete Tregs. Thus, the combination of Gem and DC^FS vaccine might alleviate the immunosuppressive effect of Tregs, thereby enhancing the treatment efficacy. We evaluated the therapeutic efficacy of the combined treatment (DC^FS + Gem) in an established Panc02 murine model. We found that the combined treatment further inhibited tumor growth, achieving a tumor inhibition rate of 89% (Fig. 4A, Supplementary Fig. 2A). When using Gem alone for treatment, a significant decrease in body weight was observed in mice, whereas this phenomenon was not seen with combined treatment (Fig. 4B). The survival period of the combined treatment was significantly prolonged, with 44% of the mice remaining alive by the 60th day (Fig. 4C). In spleen, we observed a significant increase in the number of CD8⁺ T cells in the combined treatment group. (Fig. 4D). In tumor, the use of Gem led to a significant decrease in infiltrating Tregs in the combined treatment group (Fig. 4E), and there was also a significant reduction in CD45⁺ immune cells, CD8⁺ T cells, and CD4⁺ T cells (Fig. 4F-G), but they were still significantly higher than that in other groups. The PD1⁺CD8⁺ T cells increased (Supplementary Fig. 2B). The NK cells and activated DCs in the combined treatment group significantly increased, while MDSCs showed no significant changes (Fig. 4H-J).

Immune memory is crucial for the long-term effectiveness of tumor treatment. 30 days after the completion of the DC^FS vaccine treatment, we evaluated the immune memory levels of each group. The combined treatment group exhibited significantly stronger immune memory, with a higher number of CD8⁺ T cells after re-stimulation with the antigen protein compared to the other groups (Fig. 4K-L).

3.5 Low-dose Gem can simultaneously induce immunogenic cell death to cause antigen spreading.

Low-dose Gem can also eliminate tumor cells by inducing immunogenic cell death (ICD)[20]. We detected that the Gem group and the combined treatment group induced ICD (Fig. 5A). We then tested two antigens that are highly expressed in the Panc02 pancreatic cancer tumors in mice but are lowly expressed in the normal pancreas tissue, and are not related to the vaccine (Fig. 5B). We found that the combined treatment induced a significant antigen-specific T cell response to these two antigens (Fig. 5C). This indicated that Gem caused antigen spreading through ICD, and synergized with our vaccine.

3.6 Combined treatment promoted the expression of genes related to the recruitment and activation of CD8 ⁺ T cells.

To provide a detailed characterization of the combined treatment, we conducted RNA-sequencing on tumor tissues obtained from the combined treatment group and the blank control group. A significant number of genes (768 in combined treatment group and 434 in blank control group) exhibited differential expression in both groups (Fig. 6A). To characterize the transcriptomic changes between the two groups, differentially expressed genes (DEGs) were displayed using a volcano plot (Fig. 6B) and expression heatmap (Fig. 6C). The genes Ccl17, Ccl22, and Pla2g2d, which are associated with lymphocyte infiltration, were significantly increased after the combined treatment[21–23]. Additionally, the genes related to T cell activation and cytotoxic function, including CD28, Tnfrsf4, and Cd40lg, were significantly upregulated[24–26] (Fig. 6B-C). This is consistent with the results of flow cytometry analysis. Compared to the control group, combined treatment significantly increased the infiltration of CD8⁺ T cells and also enhanced the ability of T cells to produce IFN-γ (Fig. 4G; Fig. 5C). In contrast, combination treatment suppressed the expression of tumor invasion-related genes Trp1 and Trp2 (Fig. 6B-C). Furthermore, the GO analysis of the DEGs showed that the biological processes related to lymphocyte migration as well as T cell activation and cytotoxicity were upregulated (Fig. 6D). The KEGG analysis also yielded similar results, showing that combined treatment significantly upregulated the cytokine-cytokine receptor interaction and chemokine signaling pathway (Fig. 6E). qRT-PCR was employed to validate the transcriptome results and the obtained trends were basically consistent (Fig. 6F-G). These results indicated that combined treatment promoted the infiltration of CD8⁺ T cells into tumors, stimulated their proliferation and activation, and induced the production of cytokines to eliminate tumor cells.

DCs-mediated antigen presentation plays an important role in immune regulation. Dysregulation of antigen processing and presentation is a crucial mechanism for tumors to evade immune system detection. According to reports, DCs and certain molecules involved in antigen presentation, such as TAP and HLA-Ⅰ, which are significantly reduced in pancreatic cancer[27]. In contrast to conventional vaccines, DC vaccines possess the capability to directly activate T cells. This mechanism leads to a substantial enhancement of the antigen-specific T cell response[4]. DC vaccines have demonstrated significant anti-tumor effects in various types of cancer[28–30]. Previous studies mainly used mRNA to load antigens[31, 32]. Using circular RNA to load antigens is a promising method because, compared to linear mRNA, the inherent stability of circular RNA prolongs the duration of protein translation and increases the amount of protein production[6, 7]. This helps retain antigens in DCs, prolonging antigen presentation, resulting in stronger antigen-specific CD8⁺ T cell responses and longer-lasting immune memory[33]. In this study, we successfully constructed the DC^FS vaccine and compared it with the sPD1/FS vaccine, which is another DC-based treatment method that targets antigens to DC in vivo through sPD1 to induce tumor-specific immune responses. The experimental results indicate that the efficacy of the DC^FS vaccine is significantly superior to that of the sPD1/FS vaccine. This is because sPD1/FS, as an exogenous antigen, requires cross-presentation to stimulate the production of antigen-specific CD8⁺ T cells. However, the number of cDC1s essential for cross-presentation is limited, and most antigens stimulate CD4⁺ T cells through the regular exogenous antigen presentation pathway[34–36].

Although DC vaccines can induce antigen-specific T cells effectively, their effectiveness is still limited, mainly due to the immunosuppressive TME and the tumor antigenic modulation[2, 5, 12]. We detected a significant increase in Tregs within the tumor after DC^FS vaccine treatment. Meanwhile, the levels of IFN-γ did not increase significantly as in the spleen. The increase of Tregs in the tumor suppresses the function of CD8⁺ T cells, leading to a decrease in the level of IFN-γ secretion by CD8⁺ T cells. Our DC vaccine only targets two TAAs, FAPα and survivin. Tumor cells can downregulate or even lose the targeted antigen expression, affecting the recognition of antigen-specific T cells, to reduce the therapeutic efficacy of our vaccine[37, 38]. Therefore, overcoming the inhibitory effects of the TME and promoting the release of TAAs are effective strategies for improving the therapeutic effect of DC vaccines.

For a long time, Gem, which was considered the primary treatment for pancreatic cancer, was believed to be contradictory when combined with immune-activating therapies like vaccination as it is an immunosuppressive therapy. However, mounting evidence suggests that Gem does not result in the loss of DCs or T cell function; instead, it plays a role in immune modulation by inhibiting Th2-type responses and enhancing Th1-type immune responses[39, 40]. Low-dose Gem can selectively eliminate Tregs while inducing ICD, which can eliminate tumor cells, and release antigens in situ[17, 41]. Through flow cytometry and RNA-seq analysis, it was demonstrated that combination therapy eliminated immunosuppressive factors, converting “cold” tumors into “hot” tumors. Specifically, Gem altered the immunosuppressive TME, resulting in an increase in intratumoral chemokines, enabling vaccine-induced antigen-specific T cells to infiltrate the tumor. Although it had some impact on T cell numbers, it did not affect their function. After reducing Tregs, the inhibitory effect on CD8⁺ T cells was lifted, and their activation and cytotoxic functions were further amplified. Simultaneously, Gem-induced ICD released antigens, compensating for the vaccine's limitation of targeting only two antigens, and synergized with our vaccine. DCs took up the antigens and were activated, eliciting a response from T cells specific to other antigens.

The increase in the DC vaccine migration rate is associated with a prolonged survival period. Most DC vaccines may not migrate to the lymph nodes to stimulate T cells. Efforts have been made to enhance migration rates by altering the administration route, but the optimal administration route has not yet been confirmed[42]. Even when administered directly into the lymph nodes, the immune response elicited is similar to or even not as effective as other administration routes[42, 43]. Pre-inducing a local inflammatory response at the injection site of the DC vaccine may improve the migration of DCs. Merad M et al. indicated that the local application of TLR7 agonists can enhance DC migration[44]. Meanwhile, Mitchell et al. demonstrated that pre-vaccination with tetanus/diphtheria toxoid vaccine at the injection site can also increase the lymph node migration of DC vaccines by inducing CCL3 levels[45]. Therefore, in future research, we will search for a stimulant that can induce an inflammatory response at the injection site before DC vaccination. This will promote the migration of DCs to the lymph nodes, aiming to further enhance the effectiveness of the combined treatment.

In summary, we constructed a circRNA-loaded DC vaccine and demonstrated its therapeutic efficacy in the Panc02 pancreatic tumor model. Considering the limitations of the DC vaccines, we combined it with Gem to eliminate immunosuppressive Tregs, while also inducing ICD to promote antigen spreading. The anti-tumor effect has been further improved. Our research offers a promising strategy for the clinical treatment of pancreatic cancer and establishes a foundation for the clinical trial of the DC^FS+Gem combination therapy.

Competing Interests

The authors declared no competing interests.

Ethics approval

All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Jilin University and were approved by the Institutional Animal Care and Use Committee (2023YNPZSY0913).

Consent to publish

All authors approved the fnal manuscript and its submission to this journal.

Funding

This study was supported by the Key R & D Projects of Science and Technology Department of Jilin Province [no.20230101167JC, 20180201001YY]; Jilin Provincial Department of Education Scientific Research Project [JJKH20231142KJ]; Major Projects of Science and Technology Innovation in Changchun City [no.17YJ002]; and the National Science and Technology Major Project of the Ministry of Science and Technology of China [no.2014ZX09304314-001].

Author Contribution

Z.C: substantial contributions to the conception, design, investigation, interpretation of the data and drafting of the work. Q.W, Y.S, X.Q, H.H, S.C: substantial contributions to investigation. H.W, J.W, C.W, X.Y, W.K: substantial contributions to analysis and revising the work critically for important intellectual content. H.Z: agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved, substantial contributions to the conception and evising the work critically for important intellectual content.

Acknowledgement

The authors thank Xianghua Zuo for her help in article writing.

Data Availability

The datasets used and/or analysis during the current study are available from the corresponding author on reasonable request.

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CircRNA-loaded DC vaccine in combination with low-dose gemcitabine induced potent anti-tumor immunity in pancreatic cancer model

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Cell lines, mice, and tumor model

2.2 Sources of plasmids

2.3 Preparation and Purification of Circular RNA

2.4 Generation of BMDCs in vitro

2.5 Construction of DC vaccine

2.6 In vivo immune strategies

2.7 IFN-γ ELISpot assay

2.8 Cell staining and flow cytometry

2.9 RNAseq

2.10 Statistical analysis

3. Result

3.1 Construction of circRNA-loaded DC vaccine

3.2 Immunogenicity of circRNA-loaded DC vaccine

3.3 circRNA-loaded DC vaccination had a better therapeutic effect on Panc02 pancreatic cancer

3.4 Combining low-dose Gem further enhanced the anti-tumor effect.

3.5 Low-dose Gem can simultaneously induce immunogenic cell death to cause antigen spreading.

4. Discussion

Declarations

Competing Interests

Ethics approval

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

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