Specific medicinal plant polysaccharides effectively enhance the potency of a DC-based vaccine against mouse mammary tumor metastasis

Abstract

Dendritic cell (DC) vaccines are a newly emerging immunotherapeutic approach for the treatment and prevention of cancer, but major challenges still remain particularly with respect to clinical efficacy. Engineering and optimization of adjuvant formulations for DC-based vaccines is one strategy through which more efficacious treatments may be obtained. In this study, we developed a new ex vivo approach for DC vaccine preparation. We evaluated two highly purified mixed polysaccharide fractions from the root of Astragalus membranaceus and Codonopsis pilosulae, named Am and Cp, for their use in enhancing the efficiency of a DC-based cancer vaccine against metastasis of 4T1 mammary carcinoma in mice. Mixed lymphocyte reaction showed all Am-, Cp- and [Am+Cp]-treated DCs enhanced mouse CD4+ and CD8+ T-cell proliferation. [Am+Cp]-treated DCs exhibited the strongest anti-4T1 metastasis activity in test mice. Treatments with Am, Cp and [Am+Cp] also resulted in augmented expression of CD40, CD80 and CD86 markers in test DCs. Bioinformatics analysis of the cytokine array data from treated DCs identified that [Am+Cp] is efficacious in activation of specific immune functions via mediating the expression of cytokines/chemokines involved in the recruitment and differentiation of defined immune cells. Biochemical analysis revealed that Am and Cp are composed mainly of polysaccharides containing a high level (70-95%) glucose residues, but few or no (< 1%) mannose residues. In summary, our findings suggest that the specific plant polysaccharides Am and Cp extracted from traditional Chinese medicines can be effectively used instead of bacterial LPS as a potent adjuvant in the formulation of a DC-based vaccine for cancer immunotherapies.

Fig 1. Effect of candidate adjuvants and tumor cell lysate on cell viability and T-cell proliferation.

(A) Effect of candidate adjuvants (i.e., Cp, Am and [Am+Cp]), and (B) tumor cell lysate on viability of dendritic cells. DCs (2 × 10⁵) were treated with Cp, Am or [Am+Cp] at a dose between 1 to 1000 μg/ml or treated with TCL at concentrations between 50 and 500 μg/ml for 24 h. Cell viability was performed by MTT assay. Data represent the mean ± SD of three replicates. Optimal dosage/concentration of TCL, Cp, Am and [Am+Cp] phytoextracts for stimulating DC-mediated activation of splenocyte or T-cell proliferation. (C) DCs as stimulator cells were pulsed with TCLs at 50–500 μg/ml in medium supplemented with LPS at 1 μg/ml. (D) and (E), TCL-loaded DCs as stimulator cells were treated with Cp, Am or [Am+Cp] phytoextracts at 100 or 200 μg/ml, or with 1 μg/ml LPS (positive control). The (C), (D) and (E) sets of DC stimulator cells were then co-cultured with splenocytes (C), CD8⁺ T cells (D) or CD4⁺ T cells (E), as responder cells, for 4 days. Cell proliferation activities of (C) splenocytes, (D) CD8⁺ cell and (E) CD4⁺ cells are represented as the fold change over control (i.e., splenocytes or T cells only). Data represent the mean ± SE obtained from three independent experiments. A P value of less than 0.05 was considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, no significance).

Fig 2. Adjuvant effect of [Am+Cp] phytoextract on DC vaccine against in vivo metastasis of 4T1 mammary tumors.

(A) Treatment schema used for the cancer vaccine experiment. (B) Percentage of whole body organs free from metastasis. (C) Survival rate of treated 4T1 tumor-bearing mice after resection of primary tumor(s). The free from tumor metastasis rate and mouse survival rate were statistically analyzed at day 51 after resection of the primary tumor. (D[a]) Metastatic pulmonary foci of 4T1 (shown as arrows) in lung were detected in 4T1 tumor-resected mice. (D[b]) Histological staining of tumor-bearing lung tissue sections with H&E. Arrows indicate metastatic tumors (MT: metastatic tumors; A: alveolus). (E) Bioluminescence imaging of the whole mouse body obtained with an in vivo imaging system (IVIS). (F) Immunohistochemistry staining for CD8⁺ T cells in lung tissue section. Arrows indicate the infiltrating CD8⁺ T cells in the tumor site of the lung tissues (MT: metastatic tumors).

Fig 3. Flow cytometric analysis of expression of surface markers on DCs with different treatments.

(A) Phenotypic changes in expression of CD40, CD80 and CD86 maturation markers in TCL-loaded DCs as a response to treatment with Cp, Am or [Am+Cp] phytoextracts, all at a concentration of 200 μg/ml. Anti-CD40, CD80 or CD86 antibodies were conjugated with FITC. (B) The mean fluorescence intensity (MFI) of (B[a]) CD40, (B[b]) CD80, or (B[c]) CD86 in DCs from different groups were calculated and are presented as a bar chart. Data represent the mean ± SD obtained from three independent experiments. A P value of less than 0.05 was considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, no significance).

Fig 4. Cytokine array/profiling analysis of the alteration of multiple cytokines/chemokines in conditioned culture media of Cp-, Am-, [Am+Cp]- and LPS-treated DCs.

Cytokine array membranes were incubated with cultured media from DCs that were treated with TCL for 2 h, and then with Cp, Am, [Am+Cp] or LPS for another 22 h. Squares mark the cytokines and chemokines secreted from DCs that were increased in Cp, Am, [Am+Cp] or LPS treatments: 1. CSF3, 2. CSF2, 3. CCL1, 4. IL-1α, 5. IL-1β, 6. IL-5, 7. IL-6, 8. IL-7, 9. IL-10, 10. CXCL10, 11. CXCL11, 12. CXCL1, 13. M-CSF, 14. CCL2, 15. CXCL9, 16. CCL3, 17. CCL4, 18. CXCL2, 19. TNF-α, 20. TREM-1.

Fig 5. Functional pathway and immune cell-signaling analysis of TCL-loaded DCs which were treated with Cp-, Am-, [Am+Cp]- or LPS.

The relationships between the biological functions and cytokines/chemokines of (A) DC+TCL+Cp, (B) DC+TCL+Am, (C) DC+TCL+[Am+Cp] and (D) DC+TCL+LPS were analyzed using Ingenuity Pathway Analysis (IPA) software. The cytokine/chemokine expression was determined by specific cytokine array (R&D Systems, Minneapolis, MN) and the expression levels that were altered more than two-fold in TCL groups were used in an IPA study. The signaling hubs (functions) include the differentiation of lymphocytes and dendritic cells, proliferation of helper T cells and monocytes, recruitment of phagocytes, chemotaxis of lymphocytes and inhibition of tumor growth.

Fig 6. Chromatographic separation profile of the acid hydrolysate products of Cp or Am phytoextracts as analyzed by HPAEC-PAD.

Monosaccharide composition of (A) Cp, (B) Am, and (C) mixture of seven monosaccharide standards; 1 = rhamnose (Rhm), 2 = arabinose (Ara), 3 = galactose (Gal), 4 = glucose (Glc), 5 = allose (All), 6 = galacturonic acid (GalA) and 7 = Glucuronic acid (GlcA).