Senescent cells re-engineered to express soluble programmed death receptor-1 for inhibiting programmed death receptor-1/programmed death ligand-1 as a vaccination approach against breast cancer

Abstract

Various types of vaccines have been proposed as approaches for prevention or delay of the onset of cancer by boosting the endogenous immune system. We previously developed a senescent-cell-based vaccine, induced by radiation and veliparib, as a preventive and therapeutic tool against triple-negative breast cancer. However, the programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) pathway was found to play an important role in vaccine failure. Hence, we further developed soluble programmed death receptor-1 (sPD1)-expressing senescent cells to overcome PD-L1/PD-1-mediated immune suppression while vaccinating to promote dendritic cell (DC) maturity, thereby amplifying T-cell activation. In the present study, sPD1-expressing senescent cells showed a particularly active status characterized by growth arrest and modified immunostimulatory cytokine secretion in vitro. As expected, sPD1-expressing senescent tumor cell vaccine (STCV/sPD-1) treatment attracted more mature DC and fewer exhausted-PD1⁺ T cells in vivo. During the course of the vaccine studies, we observed greater safety and efficacy for STCV/sPD-1 than for control treatments. STCV/sPD-1 pre-injections provided complete protection from 4T1 tumor challenge in mice. Additionally, the in vivo therapeutic study of mice with s.c. 4T1 tumor showed that STCV/sPD-1 vaccination delayed tumorigenesis and suppressed tumor progression at early stages. These results showed that STCV/sPD-1 effectively induced a strong antitumor immune response against cancer and suggested that it might be a potential strategy for TNBC prevention.

Keywords: immunotherapy; senescent cell; soluble PD-1; triple-negative breast cancer; vaccine.

Figure 1

Analysis of programmed death ligand‐1 (PD‐L1) expression on 4T1 cells after receiving radiation or interferon (IFN‐γ) treatment. A,B, Percentage of PD‐L1⁺ 4T1 cells was assayed by flow cytometry with the presence of different doses of IFN‐γ of 0, 5, 10, 20 and 30 ng/mL. C,D, Percentage of PD‐L1⁺ 4T1 cell was assessed on days 3 and 5 after receiving 10 Gy radiation

Figure 2

Confirmation of soluble programmed death receptor‐1 (sPD‐1) expression post‐lentivirus infection. A, Schematics of sPD‐1 overexpressing lentivirus (LV/sPD‐1) and negative control lentivirus (LV/NC). EGFP, enhanced green fluorescent protein. B, Verification of infected efficiency of lentivirus based on fluorescence microscopy of EGFP expression on 4T1 cells (magnification ×200). C, Western blotting analysis of sPD‐1 protein expression on 4T1 cells infected by LV/NC and LV/sPD‐1, with 4T1 cells as a control. D, qRT‐PCR analysis of sPD‐1 RNA expression on 4T1 cells infected by LV/NC and LV/sPD‐1, with 4T1 cells as a control. ***P < .001. E, 4T1 cells were pretreated with IFN‐γ to increase programmed death ligand‐1 expression. Culture medium from 4T1/sPD‐1 cells was added to 4T1 cells as mentioned above and incubated for approximately 30 min before flow cytometry assay for detecting ratio of PD‐1⁺ 4T1 cells. PD‐1, programmed death receptor‐1

Figure 3

Aggressive characteristics of 4T1, 4T1/NC and 4T1/sPD‐1 cells. A,B, 5‐Ethynyl‐2′‐deoxyuridine (EdU) assay was carried out to assess proliferation ability among 4T1, 4T1/NC and 4T1/sPD‐1 cells. NC, negative control; sPD‐1, soluble programmed death receptor‐1. C,D, Transwell assay was conducted to compare invasion ability among 4T1, 4T1/NC and 4T1/sPD‐1 cells at 12 h. E,F, Wound healing assay was used to assess migration ability among 4T1, 4T1/NC and 4T1/sPD‐1 cells at 12 and 24 h (magnification ×200) (ns, P > .05)

Figure 4

Senescence‐associated secretory phenotype of 4T1, 4T1/NC, and 4T1/sPD‐1 cells treated with radiation and veliparib. NC, negative control; sPD‐1, soluble programmed death receptor‐1. A, SA‐β‐gal staining was used to detect senescence. Bright blue cells were regarded as senescent (magnification ×200). B, Western blotting analysis of senescence marker p21 protein expression. C‐J, Secretion of interferon (IFN)‐γ, tumor necrosis factor‐α (TNF)‐α, interleukin (IL)‐17, IL‐12P70, transforming growth factor‐β (TGF‐β), IL‐12P40, IL‐10 and sPD‐1 was compared between non‐senescent and senescent cells (ns, non‐significant, *P < .05,**P < .01,***P < .001)

Figure 5

Tumor burden and prognosis of mice with s.c. tumor of 4T1, 4T1/NC and 4T1/sPD‐1 cells. NC, negative control; sPD‐1, soluble programmed death receptor‐1. Mice were inoculated with 4T1, 4T1/NC or 4T1/sPD‐1 cells as indicated and PBS injection was used as a control. A‐D, Individual tumor growth curves (gray lines) and mean tumor growth (black line) of mice in different groups are shown. E, Survival curves of mice in different groups

Figure 6

Analysis of dendritic cells (DC) and T‐cell subsets after vaccinations. To evaluate the effect of STCV/4T1, STCV/NC and STCV/sPD‐1, splenocytes and blood were collected from each experimental group on day 5 after vaccination. DC of splenocytes and T‐cell subsets of blood were assayed by flow cytometry. A,B, Proportions of mature DC (CD11c⁺ CD83⁺, CD11c⁺ CD86⁺) in splenocytes were analyzed by flow cytometry. Typical data from a representative experiment in mice of each group. C,D, Proportions of T‐cell subsets (PD‐1⁺ CD4⁺, PD‐1⁺ CD8⁺) in blood were analyzed by flow cytometry. Typical data from a representative experiment in mice of each group. NC, negative control; sPD‐1, soluble programmed death receptor‐1; STCV, senescent tumor cell vaccine

Figure 7

Efficacy of STCV/4T1, STCV/NC and STCV/sPD‐1 on 4T1 tumor prevention. Mice were injected with STCV/4T1, STCV/NC, or STCV/sPD‐1 on days 3 and 5 twice before 4T1 tumor cells were challenged as indicated. A‐D, Individual tumor growth (gray lines) and mean tumor growth (black line) in groups of PBS, STCV/4T1, STCV/NC, and STCV/sPD‐1 treatment are shown. E, Tumor‐free curves of mice in each group. F, Survival curves of mice in each group. NC, negative control; sPD‐1, soluble programmed death receptor‐1; STCV, senescent tumor cell vaccine

Figure 8

Efficacy of STCV/4T1, STCV/NC and STCV/sPD‐1 on 4T1 tumor treatment. Mice were injected with STCV/4T1, STCV/NC, or STCV/sPD‐1 on days 3 and 5 for twice after 4T1 tumor cells were challenged as indicated. A‐D, Individual tumor growth (gray lines) and mean tumor growth (black line) in groups of PBS, STCV/4T1, STCV/NC, and STCV/sPD‐1 treatment are shown. E, Tumor‐free curves of mice in each group. F, Survival curves of mice in each group. NC, negative control; sPD‐1, soluble programmed death receptor‐1; STCV, senescent tumor cell vaccine