A Target Animal Effectiveness Study on Adjuvant Peptide-Based Vaccination in Dogs with Non-Metastatic Appendicular Osteosarcoma Undergoing Amputation and Chemotherapy

Abstract

Despite efforts to develop novel treatment strategies, human and canine osteosarcomas continue to have poor prognosis and limited overall survival. The aim of this clinical trial was to test the antitumor effect and safety of multiple dermal administrations of a peptide-based anticancer vaccine in dogs with non-metastatic appendicular osteosarcoma undergoing standard of care (SOC), consisting of limb amputation and adjuvant chemotherapy. Salmonella-infected canine osteosarcoma cells were induced to release immunogenic peptides in the extracellular space via Cx43 hemichannels opening; the secretome was collected and constituted the vaccine. Dogs with non-metastatic appendicular osteosarcoma were eligible for recruitment. Following limb amputation and adjuvant carboplatin, dogs were vaccinated on a monthly basis for six times and followed up with serial thoracic radiographs. A population of dogs undergoing SOC treatment (amputation and adjuvant carboplatin) before the vaccine was available served as controls. Primary endpoints were time to metastasis (TTM) and tumor-specific survival (TSS). Secondary endpoints were feasibility, toxicity, T-cell and humoral immune responses. A total of 20 dogs were vaccinated along with SOC and 34 received SOC only. Vaccine-specific humoral and T-cell responses were observed; their amplitude correlated with TSS. Vaccine-associated toxicity was not recorded. TTM and TSS were significantly longer in vaccinated versus unvaccinated dogs (TTM: 308 vs. 240 days, respectively; p = 0.010; TSS: 621 vs. 278 days, respectively; p = 0.002). In dogs with non-metastatic osteosarcoma undergoing SOC, the addition of a bacteria-based vaccination strategy increased TTM, thereby prolonging survival, while maintaining a safe profile. Additionally, vaccinated dogs developed a long-term tumor-specific response, as documented by the immunomonitoring of these patients over time. These results hold promise for future management of canine osteosarcoma.

Keywords: cancer vaccine; dog; osteosarcoma; translational research.

Figure 1

Vaccine quality control. OSA primary tumor cells were infected with Salmonella or left untreated. (A). 4 h after infection, cells were fixed for immunofluorescence (IF) analysis. Cell structure was marked with alpha-SMA antibody (red), Salmonella with LPS-specific antibody (white), and hemichannels with Cx43 antibody (green). (B). ATP accumulated in cells supernatant was measured after infection. (n = 12) (C). Frequency of Annexin-PI- (live), Annexin+PI- (early apoptotic), and Annexin+PI+(apoptotic) tumor cells Salmonella-infected (Salm) or untreated (ut) (n = 2). Data are represented as mean ± SEM using a scatter dot plot. Statistical analysis was evaluated using two-sided Mann–Whitney test ** p < 0.01, **** p < 0.0001. scale bar: 10 µm and magnification: 63×.

Figure 2

Vaccinated OSA patients developed a long-term vaccine specific antitumor immune response. (A)—ELISA quantification of IFNγ released by dog patients PBMCs before vaccination (t0) and at the last vaccination (t-last) upon stimulation with vaccine (Vax). Data are shown as normalized on t0. (B)—ELISA quantification of tumor-specific IgG in dog sera. Immunomonitoring experiments were performed once; each condition was tested at least in triplicate. Data are represented as mean ± SEM using a scatter dot plot. Statistical analysis was evaluated using (A) two-sided Wilcoxon test. (B) One-way ANOVA Kruskal Wallis multiple comparison, * p < 0.05, *** p < 0.001, **** p < 0.0001. (C) Correlation Graph: Immune response level and survival.

Figure 3

(A) Time to metastasis in 54 dogs with non-metastatic appendicular osteosarcoma treated with amputation and chemotherapy, stratified according to whether they received (SOC+VAX, solid line) or did not receive (SOC, dashed line) adjuvant peptide-based vaccination (p = 0.010). (B) Tumor-specific survival in 54 dogs with non-metastatic appendicular osteosarcoma treated with amputation and chemotherapy, stratified according to whether they received (SOC+VAX, solid line) or did not receive (SOC, dashed line) adjuvant peptide-based vaccination (p = 0.002). (C) Correlation graph between TTM and TSS.