Synergistic Antitumor Effects of Caerin Peptides and Dendritic Cell Vaccines in a 4T-1 Murine Breast Cancer Model

Abstract

Background/Objectives: Breast cancer remains a leading cause of cancer-related mortality among women worldwide, necessitating novel therapeutic strategies. This study aimed to investigate the synergistic antitumor effects of caerin peptides (F1/F3) combined with dendritic cell (DC) vaccines in a 4T-1 murine breast cancer model, providing new insights for breast cancer immunotherapy. Methods: In vitro experiments evaluated the effects of F1/F3 on 4T-1 cell proliferation and apoptosis. A 4T-1 breast cancer mouse model was established, and treatments included F1/F3 alone, DC vaccines (DCV₁: loaded with whole tumor antigens; DCV₂: loaded with F1/F3-induced apoptotic antigens), or combination therapy. Flow cytometry analyzed immune cell subsets in the tumor microenvironment and lymph nodes, while ELISA measured cytokine levels. Results: F1/F3 significantly inhibited 4T-1 cell proliferation and induced apoptosis while suppressing tumor growth and lung metastasis in vivo. Flow cytometry revealed increased infiltration of CD4⁺ T cells and cDC₁ in tumors, along with reduced PD-L1 expression. DCV₂ exhibited stronger T-cell proliferation induction and lower IL-10 secretion in vitro. Combination therapy with DCV₂ and F1/F3 demonstrated superior tumor suppression compared to monotherapy. Conclusions: F1/F3 enhances antitumor immunity by modulating the tumor microenvironment, and its combination with DCV₂ yields synergistic effects. This study provides experimental evidence for combination immunotherapy in breast cancer, with potential for further optimization of DC vaccine design to improve efficacy.

Keywords: 4T-1; DC vaccine; DLNs; TME; caerin 1.1/1.9.

Figure 1

F1, F3, and F1/F3 inhibit 4T-1 cell proliferation. F1, F3, and F1/F3 inhibit the proliferation of 4T-1 cells, as assessed by the MTT assay. (A) The half-maximal inhibitory concentration (IC50) of F1 alone was 9.607 μg/mL; (B) F3 alone showed an IC50 of 15.91 μg/mL; (C) the combination of F1 and F3 (F1/F3) yielded an IC50 of 7.472 μg/mL; (D) representative microscopy images of 4T-1 cells under: no treatment (UN), P3, and F1/F3 at 10 μg/mL revealing morphological changes; Red boxes: cells with changed morphology; Microscope at 20× magnification; (E) flow cytometry results comparing apoptosis in 4T-1 cells among the UN, P3, and F1/F3 groups. Left: streaming scatter plot of different groups; Right: bar chart, UN group (blue), P3 group (red), F1/F3 group (green). Data in (A–E) represent an independent experiment repeated twice. The results are expressed as mean ± SD. ns, not significant; **** p < 0.0001. Statistical analyses were performed using one-way ANOVA for (A–C) and Student’s t-test for (E).

Figure 2

F1/F3 inhibits 4T-1 tumor growth in vivo. (A) 4T-1 cells (5 × 10⁵/200 μL) were injected subcutaneously into the lateral flanks of BALB/c mice, followed by local administration of PBS, P3, or F1/F3; (B) tumor volume and (C) survival curves; (D) tumor weights were measured on Day 18. 4T-1 cells (5 × 10⁵/200 μL) were injected into the fourth mammary fat pad of BALB/c mice, PBS, P3, or F1/F3 was administered intratumorally; (E) tumor growth and (F) survival rates in this model; (G) on day 30, lungs were isolated and stained with 15% India ink, and pulmonary nodules were counted (n: number of experimental mice). Each group has 3–8 mice. Data in (A–F) represent a single independent experiment, whereas (G) pools data from two independent experiments. The results are shown as mean ± SD. ns, not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001. Statistical analyses were carried out using two-way ANOVA for (B,E), Kaplan–Meier survival analysis for (C,F), one-way ANOVA for (D), and Student’s t-test for (G).

Figure 3

F1/F3 regulating intratumoral T cell responses in 4T-1 tumor-bearing mice. Flow cytometric analysis of T cells in the tumor microenvironment (TME) of PBS-, P3-, and F1/F3-treated 4T-1 tumor-bearing mice, including (A) T cells (CD45.2⁺CD3e⁺); (B) CD4⁺ T cells (CD45⁺CD3e⁺CD4⁺); (C) CD8⁺ T cells (CD45⁺CD3e⁺CD8⁺). PBS group (blue), P3 group (purple), F1/F3 group (pink). Each experiment used 3–6 mice per group. (A–C) represent single independent experiments, each repeated once. Data are expressed as mean ± SD. ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistical analysis was carried out by Student’s t-test.

Figure 4

Modulation of intratumoral macrophages and DC subsets in 4T-1 tumor-bearing mice. Flow cytometric analysis of macrophages and DC subsets in the tumor microenvironment (TME) of PBS-, P3-, and F1/F3-treated 4T-1 tumor-bearing mice, including (A) macrophages (CD45⁺CD11b⁺F4/80⁺), M1 (CD45⁺CD11b⁺F4/80⁺Ly6C⁺) vs. M2 (CD45⁺CD11b⁺F4/80⁺Ly6C⁻); (B) DCs (CD45.2⁺CD11c⁺MHCII⁺), cDC1 (CD45.2⁺CD11c⁺MHCII⁺XCR1⁺), cDC2 (CD45.2⁺CD11c⁺MHCII⁺XCR1⁻CD11b⁺); (C) migratory cDC1 (CD45.2⁺CD11c⁺MHCII⁺XCR1⁺CD103⁺CD8a⁻), resident cDC1 (CD45.2⁺CD11c⁺MHCII⁺XCR1⁺CD103⁻CD8a⁺), migratory cDC2 (CD45.2⁺CD11c⁺MHCII⁺XCR1⁻CD11b⁺CD103⁺CD8a⁻), resident cDC2 (CD45.2⁺CD11c⁺MHCII⁺XCR1⁻CD11b⁺CD103⁻CD8a⁺). PBS group (blue), P3 group (purple), F1/F3 group (pink). Each experiment used 3–6 mice per group. (A–C) represent single independent experiments, each repeated once. Data are expressed as mean ± SD. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistical analysis was carried out by Student’s t-test.

Figure 5

F1/F3 modulates T Cells, DC subsets, and PD-1/PD-L1 expression in the lymph nodes of 4T1 tumor-bearing mice. Flow cytometry was performed on draining lymph nodes from PBS- or F1/F3-treated mice to evaluate immune cells, DC subsets, and related factors: (A) CD45.2⁺ cells; (B) T cells (CD45.2⁺CD3e⁺); (C) CD4⁺ T cells (CD45⁺CD3e⁺CD4⁺), CD8⁺ T cells (CD45⁺CD3e⁺CD8⁺); (D) cDC1 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺CD11b⁻) vs. cDC2 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺CD11b⁺); (E) PD-1 (CD45.2⁺CD3e⁺PD-1⁺); (F) PD-L1 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺PD-L1⁺); (G) PD-L1 expression in cDC1 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺CD11b⁻PD-L1⁺) and cDC2 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺CD11b⁺PD-L1⁺), as well as PD-1 on CD4⁺ T cells (CD45⁺CD3e⁺CD4⁺PD-1⁺) and CD8⁺ T cells (CD45⁺CD3e⁺CD8⁺PD-1⁺). PBS group (blue), P3 group (purple). Each experiment used 3–6 mice per group. Data in (A–G) represent single independent experiments, repeated once, shown as mean ± SD or mean ± SEM. ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistical analysis was conducted by Student’s t-test.

Figure 6

F1/F3 improves the immune profile in non-draining lymph nodes of 4T-1 tumor-bearing Mice. Flow cytometric analysis was performed on non-draining lymph nodes from PBS- or F1/F3-treated mice to examine immune cells, DC subsets, and related markers, including (A) CD45.2⁺ cells; (B) T cells (CD45.2⁺CD3e⁺); (C) CD4⁺ T cells (CD45⁺CD3e⁺CD4⁺), CD8⁺ T cells (CD45⁺CD3e⁺CD8⁺); (D) cDC1 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺CD11b⁻) vs. cDC2 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺CD11b⁺); (E) PD-1 (CD45.2⁺CD3e⁺PD-1⁺); (F) PD-L1 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺PD-L1⁺), and (G) PD-L1 expression on cDC1 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺CD11b⁻PD-L1⁺), cDC2 (CD45.2⁺Lineage⁻CD11c⁺MHCII⁺CD11b⁺PD-L1⁺), as well as PD-1 on CD4⁺ T cells (CD45⁺CD3e⁺CD4⁺PD-1⁺) and CD8⁺ T cells (CD45⁺CD3e⁺CD8⁺PD-1⁺). PBS group (blue), P3 group (purple). Each experiment included 3–6 mice per group. Data in (A–G) represent a single independent experiment, repeated once. The results are shown as mean ± SD. ns: not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Student’s t-test was performed (A–G).

Figure 7

DC vaccine inhibits 4T-1 tumor growth in vivo. (A) A 4T-1 tumor-bearing mouse model was established in the lateral flank. Tumor growth (B) and survival rates (C) were compared among the untreated (UN) group, DC vaccine group (DCV), and F1/F3 group; flow cytometric analysis of draining lymph nodes measured; (D) IFN-γ secretion (CD45⁺CD3e⁺CD8⁺IFN-γ⁺) and T-cell subsets, including (E) CD45.2⁺ cells, T cells (CD45.2⁺CD3e⁺), CD4⁺ T cells (CD45⁺CD3e⁺CD4⁺), and CD8⁺ T cells (CD45⁺CD3e⁺CD8⁺); UN group (blue), DCV group (red). each group contained 3–6 mice. Data in (A–E) represent a single independent experiment repeated once, shown as mean ± SD. Two-way ANOVA was used for (B), Kaplan–Meier analysis for (C), and Student’s t-test for (D,E). ns, not significant; ** p < 0.01; **** p < 0.0001.

Figure 8

Immunomodulatory effects of DC vaccine on intratumoral T cells in 4T-1 tumor-bearing mice. Flow cytometric analysis of T cell subsets in the draining lymph nodes of the untreated (UN) group and DC vaccine group (DCV), including (A) effector T cells (CD45.2⁺CD3e⁺CD44highCD62L⁻), effector CD4⁺ T cells (CD45.2⁺CD3e⁺CD44highCD62L⁻CD4⁺), effector CD8⁺ T cells (CD45.2⁺CD3e⁺CD44highCD62L⁻CD8⁺); (B) naïve T cells (CD45.2⁺CD3e⁺CD62LhighCD44⁻), naïve CD4⁺ T cells (CD45.2⁺CD3e⁺CD62LhighCD44⁻CD4⁺), and naïve CD8⁺ T cells (CD45.2⁺CD3e⁺CD62LhighCD44⁻CD8⁺); (C) effector memory T cells (CD45.2⁺CD3e⁺CD44highCD62L⁻CCR7⁻), effector memory CD4⁺ T cells (CD45.2⁺CD3e⁺CD44highCD62L⁻CCR7⁻CD4⁺), and effector memory CD8⁺ T cells (CD45.2⁺CD3e⁺CD44highCD62L⁻CCR7⁻CD8⁺). UN group (blue), DCV group (red). Each group contained 3–6 mice. Data in (A–C) represent a single independent experiment repeated once, shown as mean ± SD. ns, not significant; *** p < 0.001; **** p < 0.0001. Statistical analysis was conducted by Student’s t-test.

Figure 9

DCV₂ exhibits superior antitumor activity compared to dCv₁. (A) The 4T-1 tumor-bearing mouse model was established in the lateral flank. Mice received either DCV₁, DCV₂, or F1/F3 alone, and (B) tumor growth and (C) survival rates were recorded. Next, to determine whether DC vaccines could enhance the efficacy of F1/F3, the mice were treated with DCV₁ + F1/F3 or DCV₂ + F1/F3 (As shown in (A)), and (D) tumor growth was measured. Each group contained 3–6 mice. Data in (A–D) represent single independent experiments, each repeated once. The results are shown as mean ± SD. ns, not significant; ** p < 0.01, **** p < 0.0001. Statistical significance was evaluated by two-way ANOVA for (B,D) and Kaplan–Meier survival analysis for (C).

Figure 10

In vitro co-culture of DCV₁ or DCV₂ with T Cells and tumor cells. Flow cytometric analysis shows (A) that both DCV₁ and DCV₂ enhance T cell-mediated cytotoxicity against tumor cells (4T-1/TC-1) (DCV₁ + T + Tumor (blue), DCV₂ + T + Tumor (red), Tumor (green), T + Tumor (purple)) and (B) promote T-cell proliferation(DCV₁ + T+Tumor (blue), DCV₂ + T + Tumor (red), T + Tumor (green)). Using ELISA kits, we quantified immune-related cytokines in the co-culture supernatants, including (C) TNF-α, (DCV₁ + T + Tumor (purple), DCV₂ + T + Tumor (blue), Tumor (green), T + Tumor (Orange)) (D) IL-12, and (E) IL-10 (DCV₁ + T + Tumor (blue), DCV₂ + T + Tumor (purple), Tumor (pink), T + Tumor (green)). Data in (A–E) represent a single independent experiment repeated once, shown as mean ± SD. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistical analysis was conducted by Student’s t-test for (A,B) or one-way ANOVA for (C–E).