Low-dose decitabine enhances the efficacy of viral cancer vaccines for immunotherapy

Abstract

Cancer immunotherapy requires a specific antitumor CD8⁺ T cell-driven immune response; however, upon genetic and epigenetic alterations of the antigen processing and presenting components, cancer cells escape the CD8⁺ T cell recognition. As a result, poorly immunogenic tumors are refractory to conventional immunotherapy. In this context, the use of viral cancer vaccines in combination with hypomethylating agents represents a promising strategy to prevent cancer from escaping immune system recognition. In this study, we evaluated the sensitivity of melanoma (B16-expressing ovalbumin) and metastatic triple-negative breast cancer (4T1) cell lines to FDA-approved low-dose decitabine in combination with PeptiCRAd, an adenoviral anticancer vaccine. The two models showed different sensitivity to decitabine in vitro and in vivo when combined with PeptiCRAd. In particular, mice bearing syngeneic 4T1 cancer showed higher tumor growth control when receiving the combinatorial treatment compared to single controls in association with a higher expression of MHC class I on cancer cells and reduction in Tregs within the tumor microenvironment. Furthermore, remodeling of the CD8⁺ T cell infiltration and cytotoxic activity toward cancer cells confirmed the effect of decitabine in enhancing anticancer vaccines in immunotherapy regimens.

Keywords: MT: Regular Issue; cancer vaccines; epigenetic therapy; immunotherapy; oncolytic vaccines; oncolytic viruses.

Graphical abstract

Figure 1

Expression level of MHC-I and PD-L1 in melanoma (human, A-375; murine B16-OVA) and TNBC (human, MDA-MB-436; murine, 4T1) cell lines (A) The cells were stimulated with 0.1 μM (low dose) or 1 μM (high dose) DAC for 4 days, followed by the addition of IFN-γ as shown in schematic (A). (B–I) MFI of MHC class I (B–E) and PD-L1 (F–I) were then analyzed by flow cytometry using anti-mouse or anti-human fluorophore-conjugated antibodies. N = 3 in (B)–(I). Levels of significance were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 (2-way ANOVA with Tukey’s multiple comparisons correction to compare individual groups). Graphs are shown as mean ± SD.

Figure 2

Cell viability in in melanoma (human, A-375; murine B16-OVA) and TNBC (human, MDA-MB-436; murine, 4T1) cell lines (A) The cells were stimulated with 0.1 μM (low dose) or 1 μM (high dose) of DAC for 4 days and followed by the infection with Ad-5/3 Δ24 (Ad). (B–I) Cell viability assay was evaluated using MTS assay. The cells were infected with Ad5/3Δ24 (Ad) (black), Ad + DAC 0.1 μM (purple), or Ad + DAC 1 μM (pink) at different MOIs, as indicated in (B), (D), (F), and (G). (C), (E), (H), and (I) represent cell viability after different doses of DAC (0.1 and 1 μM). N = 4. Levels of significance were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 (2-way ANOVA with Tukey’s multiple comparisons correction to compare individual groups). Graphs are shown as mean ± SD.

Figure 3

Tumor growth in syngeneic mouse model B16-OVA and 4T1 Treatment schematics are depicted in (A) and (E) for B16-OVA and 4T1, respectively. Normalized tumor growth curves for each model are shown in (B) and (F); the number of mice in the B16-OVA model was 5 in the MOCK group and 7 in the others; the number of mice in the 4T1 model was 8–10. Tumor volumes were normalized on day 4 measurements. (C) and (G) show the volumes that the tumors from each treatment reached at day 28 in B16-OVA and 4T1, respectively. The graphs in (D) and (H) show single tumor growth curves for each treatment group. Error bars represent SEMs. Levels of significance were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 (2-way ANOVA with Tukey’s multiple comparisons correction to compare individual groups).

Figure 4

Intratumoral expression of MHC class I and PD-L1 and lymphocyte infiltration Intratumoral expression MHC-I (A and G), PDL-1 (B and H), CD4 (C and I), Tregs (D and J), CD8 (E and K), and exhaustion markers (PD-1, TIM-3). (F and L) Exhaustion markers were evaluated within the TME in B16-OVA and 4T1. N = 5; each dot represents a single mouse. Horizontal bar represents the mean. Levels of significance were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 (2-way ANOVA with Tukey’s multiple comparisons correction to compare individual groups).

Figure 5

Multiplex IHC of 4T1 tumors Representative images from the 4T1 murine tumors were antibody stained with lymphocyte (CD8), endothelial (CD31), and tumor (pan-CK and E-cadherin) markers (A). In addition, all of the histological sections were counterstained with H&E. Graphs are shown as mean ± SD. N = 10 pictures were analyzed from 3 different tumors. The histograms (B–D) show the mean value for each signal area percentage. Levels of significance were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 (1-way ANOVA with Tukey’s multiple comparisons correction to compare individual groups). (E–H) show a whole-tumor section stained with CD8 and pan-CK/E-cadherin. Representative regions for CD8 and CD31 staining are displayed for each tumor section from each treatment group (bar, 100 μm). (I) represents the DAPI staining of the tumor sections (bar, 500 μm).

Figure 6

ELISpot IFN-γ analysis was performed on splenocytes harvested from B16-OVA or 4T1-bearing mice (A and B) In B16-OVA, splenocytes reactivity was tested against Ad-5/3 Δ24 (A) and OVA (SIINFEKL) (B). (C) The frequencies of T cell responses are depicted as spot-forming units per 1 × 10⁶ splenocytes; the amount of antitumor CD8⁺ TILs was measured by flow cytometry using APC-conjugated H-2Kb-SIINFEKL pentamer staining; each dot represents a single mouse; the mean is represented by the horizontal bar. (D and E) In 4T1, splenocytes reactivity was tested against Ad-5/3 Δ24 (D) and ERV2 (TYVAGDTQV) (E). Fresh splenocytes were harvested from five 4T1-bearing mice, and combined and cocultured with 4T1 cells previously treated with low-dose DAC. LDH release assay was performed on culture supernatant after 72 h. N = 5 in (A–E). N = 5 mice splenocytes were combined and analyzed in technical triplicates in (F). Levels of significance were set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 (1-way ANOVA with Tukey’s multiple comparisons correction to compare individual groups). Graphs are shown as mean ± SEM.