Antitumor efficacy of a neoantigen cancer vaccine delivered by electroporation is influenced by microbiota composition

Abstract

Cancer is a heterogeneous disease and its treatment remains unsatisfactory with inconstant therapeutic responses. This variability could be related, at least in part, to different and highly personalized gut microbiota compositions. Different studies have shown an impact of microbiota on antitumor therapy. It has been demonstrated that some gut bacteria influences the development and differentiation of immune cells, suggesting that different microbiota compositions could affect the efficacy of the antitumor vaccine. Emerging data suggest that recognition of neoantigens for the generation of neoantigen cancer vaccines (NCVs) could have a key role in the activity of clinical immunotherapies. However, it is still unknown whether there is a crosstalk between microbiota and NCV. This study aimed to understand the possible mechanisms of interaction between gut microbiota and NCV delivered by DNA-electroporation (DNA-EP). We found that decreased microbiota diversity induced by prolonged antibiotic (ATB) treatment is associated with higher intratumor specific immune responses and consequently to a better antitumor effect induced by NCV delivered by DNA-EP.

Keywords: Microbiota; T cells; neoantigens; vaccine.

Figure 1.

ATB treatment enables NCV to delay tumor growth. C57Bl/6 mice were treated or not with ATB starting from day zero for the whole experiment, vaccinated with M2 DNA-EP (see M&M) once a week for four weeks, and injected with MC38 cells on day 45. a, Tumor volumes were measured twice a week by caliper. Tumor growth was significantly reduced in vaccinated and ATB treated mice as compared to the control group (* p < 0.05) and vaccinated mice (** p < 0.01). Unpaired two-tailed Student’s t-tests were conducted. b, Percentage of tumor-free mice. Sixteen mice per group were utilized in three independent experiments. The results of one representative experiment out of three are shown. The tumor-bearing mice curve of DNA-EP and ATB treated mice is significantly different (p < 0.0298) compared to the DNA-EP group. Log-rank (Mantel-Cox) test was conducted

Figure 2.

ATB treatment affects neoantigen-specific immune responses induced by DNA-EP. a, b, Immune response was evaluated one week after the last vaccination on day 43 (a, b) and one week after tumor challenge on day 52 (c) by flow cytometry in the PBMCs. To analyze neoantigen-specific cytokine production by intracellular staining live cells were gated on CD3⁺CD8⁺ for IFNγ and TNFα production. Unpaired two-tailed Student’s t-tests were conducted (*** p < 0.0001). c, At the end of the experiment on day 65 mice were sacrificed and IFNγ producing cells were evaluated by IFNγ ELIspot assay with splenocytes restimulated with neoantigens pool (e, f). Unpaired two-tailed Student’s t-tests were conducted. Data are from two independent experiments

Figure 3.

Vaccination increases the diversity reduced by ATB administration. Stool samples were analyzed before the tumor challenge on day 44. a, Bacteria load was expressed by operational taxonomic unit (OTU)/mg of stools and measured by NGS 16 S. b, Principal component (PC) analysis between groups. The PC1 explains 42,3% of the total variance, PC2 explains 8,7%. c, Alpha diversity, measured by Shannon’s index, represents species richness. d, Beta diversity represents the different microbiota composition between two groups. The value can be from 0 to 1, where 0 represents groups with similar species composition and 1 represents groups with no species in common. In the beta diversity, a weighted average was considered. Data are from two independent experiments

Figure 4.

SCFAs metabolites do not correlate with tumor growth. Level of SCFAs analyzed in the serum of mice one week after tumor challenge on day 52 determined using LC-SIM-MS (n = 5 mice for group). Data are from one out of two experiments. Unpaired two-tailed Student’s t-tests were conducted (*p < 0.05)

Figure 5.

Increased TIL effector functions in DNA-EP and ATB treated mice a, Volcano plot demonstrating the significantly differentially expressed genes, highlighting Arg1 and Gzmc. b, Level of RNA expression (FPMK, fragments per kilobase of exon model per million reads mapped) of Gzmc, Gzmb, and Arg1 measured by RNAseq in tumors of mice sacrificed one week after tumor challenge. c, Tumor expression of Gzmc, Gzmb, and Arg1 genes assessed by RT-PCR. d, Flow cytometry (FC) analysis of IFNγ and TNFα expression in TILs (CD4 and CD8) gated on CD45⁺CD3⁺ and stimulated with PMA/iono. e, FC analysis of cytokines expression of innate immune cells gated on CD45⁺CD3⁻CD11b⁺CD11c⁻; monocytes (Ly6C^highLy6G⁻), macrophages (Ly6C^lowLy6G⁻), and polymorphonucleated (PMN, Ly6C^highLy6G⁺). Natural killer (NK, NK1.1⁺) cells were gated on CD45⁺CD3⁻. f, FC analysis of IFNγ and TNFα expression in TILs (CD3⁺CD8⁺) gated on CD45⁺ and stimulated with M2-specific peptides. g, FC analysis of IFNγ and TNFα expression in splenocytes (CD3⁺CD8⁺) gated on CD45⁺ and stimulated with M2-specific peptides. Unpaired two-tailed Student’s t-tests were conducted. (*p < 0.05). Data are from two independent experiments

Figure 6.

Microbiota alteration elicits T cell activation in DNA-EP treated mice. A schematic model of possible crosstalk between microbiota and tumor is represented. ATB treatment, in combination with a vaccine, modifies microbiota composition reducing some bacteria species in favor of others that are not normally present in mice. This microbiota alteration induces a reduction of Arginase1 in tumor lysate, possibly related to reported TIL activation in the DNA EP+ATB group